Vascular endothelial growth factor D (VEGF-D) antibodies and vectors, and methods of use

ABSTRACT

VEGF-D, a new member of the PDGF family of growth factors, which among other things stimulates endothelial cell proliferation and angiogenesis and increases vascular permeability, as well as nucleotide sequences encoding it, methods for producing it, antibodies and other antagonists to it, transfected or transformed host cells for expressing it, pharmaceutical compositions containing it, and uses thereof in medical and diagnostic applications.

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of the filing dates of thefollowing copending U.S. Provisional Applications: Serial No.60/023,751, filed Aug. 23, 1996; Serial No. 60/031,097, filed Nov. 14,1996; Serial No. 60/038,814, filed Feb. 10, 1997; and Serial No.60/051,426, filed Jul. 1, 1997.

FIELD OF THE INVENTION

[0002] This invention relates to growth factors for endothelial cells,and in particular to a novel vascular endothelial growth factor, DNAencoding the factor, and to pharmaceutical and diagnostic compositionsand methods utilising or derived from the factor.

BACKGROUND OF THE INVENTION

[0003] Angiogenesis is a fundamental process required for normal growthand development of tissues, and involves the proliferation of newcapillaries from pre-existing blood vessels. Angiogenesis is not onlyinvolved in embryonic development and normal tissue growth, repair, andregeneration, but is also involved in the female reproductive cycle,establishment and maintenance of pregnancy, and in repair of wounds andfractures. In addition to angiogenesis which takes place in the normalindividual, angiogenic events are involved in a number of pathologicalprocesses, notably tumour growth and metastasis, and other conditions inwhich blood vessel proliferation, especially of the microvascularsystem, is increased, such as diabetic retinopathy, psoriasis andarthropathies. Inhibition of angiogenesis is useful in preventing oralleviating these pathological processes.

[0004] On the other hand, promotion of angiogenesis is desirable insituations where vascularization is to be established or extended, forexample after tissue or organ transplantation, or to stimulateestablishment of collateral circulation in tissue infarction or arterialstenosis, such as in coronary heart disease and thromboangitisobliterans.

[0005] Because of the crucial role of angiogenesis in so manyphysiological and pathological processes, factors involved in thecontrol of angiogenesis have been intensively investigated. A number ofgrowth factors have been shown to be involved in the regulation ofangiogenesis; these include fibroblast growth factors (FGFs),platelet-derived growth factor (PDGF), transforming growth factor α(TGFα), and hepatocyte growth factor (HGF). See for example Folkman etal, “Angiogenesis”, J. Biol. Chem., 1992 267 10931-10934 for a review.

[0006] It has been suggested that a particular family of endothelialcell-specific growth factors and their corresponding receptors isprimarily responsible for stimulation of endothelial cell growth anddifferentiation, and for certain functions of the differentiated cells.These factors are members of the PDGF family, and appear to act viaendothelial receptor tyrosine kinases (RTKs). Hitherto four vascularendothelial growth factor subtypes have been identified. Vascularendothelial growth factor (VEGF), now known as VEGF-A, has been isolatedfrom several sources. VEGF-A shows highly specific mitogenic activityagainst endothelial cells, and can stimulate the whole sequence ofevents leading to angiogenesis. In addition, it has strongchemoattractant activity towards monocytes, can induce plasminogenactivator and plasminogen activator inhibitor in endothelial cells, andcan also influence microvascular permeability. Because of the latteractivity, it is also sometimes referred to as vascular permeabilityfactor (VPF). The isolation and properties of VEGF have been reviewed;see Ferrara et al, “The Vascular Endothelial Growth Factor Family ofPolypeptides”, J. Cellular Biochem., 1991 47 211-218 and Connolly,“Vascular Permeability Factor: A Unique Regulator of Blood VesselFunction”, J. Cellular Biochem., 1991 47 219-223.

[0007] More recently, three further members of the VEGF family have beenidentified. These are designated VEGF-B, described in InternationalPatent Application No. PCT/US96/02957 (WO 96/26736) by Ludwig Institutefor Cancer Research and The University of Helsinki, VEGF-C, described inJoukov et al, The EMBO Journal, 1996 15 290-298, and VEGF2, described inInternational Patent Application No. PCT/US94/05291 (WO 95/24473) byHuman Genome Sciences, Inc. VEGF-B has closely similar angiogenic andother properties to those of VEGF, but is distributed and expressed intissues differently from VEGF. In particular, VEGF-B is very stronglyexpressed in heart, and only weakly in lung, whereas the reverse is thecase for VEGF. This suggests that VEGF and VEGF-B, despite the fact thatthey are co-expressed in many tissues, may have functional differences.

[0008] VEGF-B was isolated using a yeast co-hybrid interaction trapscreening technique, screening for cellular proteins which mightinteract with cellular retinoic acid-binding protein type I (CRABP-I).Its isolation and characteristics are described in detail inPCT/US96/02597 and in Olofsson et al, Proc. Natl. Acad. Sci., 1996 932576-2581.

[0009] VEGF-C was isolated from conditioned media of PC-3 prostateadenocarcinoma cell line (CRL1435) by screening for ability of themedium to produce tyrosine phosphorylation of the endothelialcell-specific receptor tyrosine kinase Flt4, using cells transfected toexpress Flt4. VEGF-C was purified using affinity chromatography withrecombinant Flt4, and was cloned from a PC-3 cDNA library. Its isolationand characteristics are described in detail in Joukov et al, The EMBOJournal, 1996 15 290-298.

[0010] VEGF2 was isolated from a highly tumorgenic,oestrogen-independent human breast cancer cell line. While this moleculeis stated to have about 22% homology to PDGF and 30% homology to VEGF,the method of isolation of the gene encoding VEGF2 is unclear, and nocharacterization of the biological activity is disclosed.

[0011] Vascular endothelial growth factors appear to act by binding toreceptor tyrosine kinases of the PDGF-receptor family. Five endothelialcell-specific receptor tyrosine kinases have been identified, namelyFlt-1 (VEGFR-1), KDR/Flk-1 (VEGFR-2), Flt4 (VEGFR-3), Tie and Tek/Tie-2.All of these have the intrinsic tyrosine kinase activity which isnecessary for signal transduction. The essential, specific role invasculogenesis and angiogenesis of Flt-1, Flk-1, Tie and Tek/Tie-2 hasbeen demonstrated by targeted mutations inactivating these receptors inmouse embryos. VEGFR-1 and VEGFR-2 bind VEGF with high affinity, andVEGFR-1 also binds VEGF-B and placenta growth factor (PlGF). VEGF-C hasbeen shown to be the ligand for Flt4 (VEGFR-3), and also activatesVEGFR-2 (Joukov et al, 1996). A ligand for Tek/Tie-2 has been described(International Patent Application No. PCT/US95/12935 (WO 96/11269) byRegeneron Pharmaceuticals, Inc.); however, the ligand for Tie has notyet been identified.

[0012] The receptor Flt-4 is expressed in venous and lymphaticendothelia in the fetus, and predominantly in lymphatic endothelia inthe adult (Kaipainen et al, Cancer Res, 1994 54 6571-6577; Proc Natl.Acad. Sci. USA, 1995 92 3566-3570). It has been suggested that VEGF-Cmay have a primary function in lymphatic endothelium, and a secondaryfunction in angiogenesis and permeability regulation which is sharedwith VEGF (Joukov et al, 1996).

[0013] We have now isolated human cDNA encoding a novel protein of thevascular endothelial growth factor family. The novel protein, designatedVEGF-D, has structural similarities to other members of this family.

SUMMARY OF THE INVENTION

[0014] The invention generally provides an isolated novel growth factorwhich has the ability to stimulate and/or enhance proliferation ordifferentiation of endothelial cells, isolated DNA sequences encodingthe novel growth factor, and compositions useful for diagnostic and/ortherapeutic applications.

[0015] According to one aspect, the invention provides an isolated andpurified nucleic acid molecule which encodes a novel polypeptide,designated VEGF-D, which is structurally homologous to VEGF, VEGF-B andVEGF-C. In a preferred embodiment, the nucleic acid molecule is a cDNAwhich comprises the sequence set out in SEQ ID NO. 1, SEQ ID NO. 4, SEQID NO. 6 or SEQ ID NO. 7. This aspect of the invention also encompassesDNA molecules of sequence such that they hybridise under stringentconditions with DNA of SEQ ID NO. 1, SEQ ID NO. 4, SEQ ID NO. 6 or SEQID NO. 7. Preferably the DNA molecule able to hybridise under stringentconditions encodes the portion of VEGF-D from amino acid residue 93 toamino acid residue 201, optionally operatively linked to a DNA sequenceencoding FLAG™ peptide.

[0016] Preferably the cDNA comprises the sequence set out in SEQ ID NO.4, SEQ ID NO. 6 or SEQ ID NO. 7, more preferably that of SEQ ID NO. 4.

[0017] According to a second aspect, the invention provides apolypeptide possessing the characteristic amino acid sequence:

[0018] Pro-Xaa-Cys-Val-Xaa-Xaa-Xaa-Arg-Cys-Xaa-Gly-Cys-Cys (SEQ ID NO.2),

[0019] said polypeptide having the ability to stimulate proliferation ofendothelial cells, and said polypeptide comprising a sequence of aminoacids substantially corresponding to the amino acid sequence set out inSEQ ID NO. 3, or a fragment or analogue thereof which has the ability tostimulate one or more of endothelial cell proliferation,differentiation, migration or survival.

[0020] These abilities are referred to herein as “biological activitiesof VEGF-D” and can readily be tested by methods known in the art.Preferably the polypeptide has the ability to stimulate endothelial cellproliferation or differentiation, including, but not limited to,proliferation or differentiation of vascular endothelial cells and/orlymphatic endothelial cells.

[0021] More preferably the polypeptide has the sequence set out in SEQID NO. 5, SEQ ID NO. 8 or SEQ ID NO. 9, and most preferably has thesequence set out in SEQ ID NO. 5.

[0022] A preferred fragment of the polypeptide invention is the portionof VEGF-D from amino acid residue 93 to amino acid residue 201,optionally linked to FLAG™ peptide. Where the fragment is linked toFLAG™, the fragment is VEGFDΔNΔC, as hereindefined.

[0023] Thus polypeptides comprising conservative substitutions,insertions, or deletions, but which still retain the biological activityof VEGF-D, are clearly to be understood to be within the scope of theinvention. The person skilled in the art will be well aware of methodswhich can readily be used to generate such polypeptides, for example theuse of site-directed mutagenesis, or specific enzymic cleavage andligation. The skilled person will also be aware that peptidomimeticcompounds or compounds in which one or more amino acid residues arereplaced by a non-naturally occurring amino acid or an amino acidanalogue may retain the required aspects of the biological activity ofVEGF-D. Such compounds can readily be made and tested by methods knownin the art, and are also within the scope of the invention.

[0024] In addition, variant forms of the VEGF-D polypeptide which resultfrom alternative splicing, as are known to occur with VEGF, andnaturally-occurring allelic variants of the nucleic acid sequenceencoding VEGF-D are encompassed within the scope of the invention.Allelic variants are well known in the art, and represent alternativeforms or a nucleic acid sequence which comprise substitution, deletionor addition of one or more nucleotides, but which do not result in anysubstantial functional alteration of the encoded polypeptide.

[0025] As used herein, the term “VEGF-D” collectively refers to thepolypeptides of SEQ ID NO. 3, SEQ ID NO. 5, SEQ ID NO. 8 and SEQ ID NO.9 and fragments or analogues thereof which have the biological activityof VEGF-D as herein defined.

[0026] Such variant forms of VEGF-D can be prepared by targetingnon-essential regions of the VEGF-D polypeptide for modification. Thesenon-essential regions are expected to fall outside thestrongly-conserved regions indicated in the figures herein, especiallyFIG. 2 and FIG. 10. In particular, the growth factors of the PDGFfamily, including VEGF, are dimeric, and VEGF-B, VEGF-C, PlGF, PDGF-Aand PDGF-B show complete conservation of 8 cysteine residues in theN-terminal domains, ie. the PDGF-like domains (Olofsson et al, 1996;Joukov et al, 1996). These cysteines are thought to be involved inintra- and inter-molecular disulphide bonding. In addition there arefurther strongly, but not completely, conserved cysteine residues in theC-terminal domains. Loops 1, 2 and 3 of each subunit, which are formedby intra-molecular disulphide bonding, are involved in binding to thereceptors for the PDGF/VEGF family of growth factors (Andersson et al:Growth Factors, 1995 12 159-164) As shown herein, the cysteinesconserved in previously known members of the VEGF family are alsoconserved in VEGF-D.

[0027] The person skilled in the art thus is well aware that thesecysteine residues should be preserved in any proposed variant form, andthat the active sites present in loops 1, 2 and 3 also should bepreserved. However, other regions of the molecule can be expected to beof lesser importance for biological function, and therefore offersuitable targets for modification. Modified polypeptides can readily betested for their ability to show the biological activity of VEGF-D byroutine activity assay procedures such as cell proliferation tests.

[0028] It is contemplated that some modified VEGF-D polypeptides willhave the ability to bind to endothelial cells, ie. to VEGF-D receptors,but will be unable to stimulate endothelial cell proliferation,differentiation, migration or survival. These modified polypeptides areexpected to be able to act as competitive or non-competitive inhibitorsof VEGF-D, and to be useful in situations where prevention or reductionof VEGF-D action is desirable. Thus such receptor-binding butnon-mitogenic, non-differentiation inducing, non-migration inducing ornon-survival promoting variants of VEGF-D are also within the scope ofthe invention, and are referred to herein as “receptor-binding butotherwise inactive variants”.

[0029] According to a third aspect, the invention provides a purifiedand isolated nucleic acid encoding a polypeptide or polypeptide fragmentof the invention. The nucleic acid may be DNA, genomic DNA, cDNA or RNA,and may be single-stranded or double stranded. The nucleic acid may beisolated from a cell or tissue source, or of recombinant or syntheticorigin. Because of the degeneracy of the genetic code, the personskilled in the art will appreciate that many such coding sequences arepossible, where each sequence encodes the amino acid sequence shown inSEQ ID NO. 3, SEQ ID NO. 5, SEQ ID NO. 8 or SEQ ID NO. 9, an activefragment or analogue thereof, or a receptor-binding but otherwiseinactive or partially inactive variant thereof.

[0030] A fourth aspect of the invention provides vectors comprising thecDNA of the invention or a nucleic acid according to the third aspect ofthe invention, and host cells transformed or transfected with nucleicacids or vectors of the invention. These cells are particularly suitablefor expression of the polypeptide of the invention, and include insectcells such as Sf9 cells, obtainable from the American Type CultureCollection (ATCC SRL-171), transformed with a baculovirus vector, andthe human embryo kidney cell line 293EBNA transfected by a suitableexpression plasmid. Preferred vectors of the invention are expressionvectors in which a nucleic acid according to the invention isoperatively connected to one or more appropriate promoters and/or othercontrol sequences, such that appropriate host cells transformed ortransfected with the vectors are capable of expressing the polypeptideof the invention. Other preferred vectors are those suitable fortransfection of mammalian cells, or for gene therapy, such as adenovirusor retrovirus vectors or liposomes. A variety of such vectors is knownin the art.

[0031] The invention also provides a method of making a vector capableof expressing a polypeptide encoded by a nucleic acid according to theinvention, comprising the steps of operatively connecting the nucleicacid to one or more appropriate promoters and/or other controlsequences, as described above.

[0032] The invention further provides a method of making a polypeptideaccording to the invention, comprising the steps of expressing a nucleicacid or vector of the invention in a host cell, and isolating thepolypeptide from the host cell or from the host cell's growth medium. Inone preferred embodiment of this aspect of the invention, the expressionvector further comprises a sequence encoding an affinity tag, such asFLAG™ or hexahistidine, in order to facilitate purification of thepolypeptide by affinity chromatography.

[0033] In yet a further aspect, the invention provides an antibodyspecifically reactive with a polypeptide of the invention. This aspectof the invention includes antibodies specific for the variant forms,fragments and analogues of VEGF-D referred to above. Such antibodies areuseful as inhibitors or agonists of VEGF-D and as diagnostic agents fordetection and quantification of VEGF-D. Polyclonal or monoclonalantibodies may be used. Monoclonal and polyclonal antibodies can beraised against polypeptides of the invention using standard methods inthe art. For some purposes, for example where a monoclonal antibody isto be used to inhibit effects of VEGF-D in a clinical situation, it maybe desirable to use humanized or chimeric monoclonal antibodies. Methodsfor producing these, including recombinant DNA methods, are also wellknown in the art.

[0034] This aspect of the invention also includes an antibody whichrecognises VEGF-D and which is suitably labelled.

[0035] Polypeptides or antibodies according to the invention may belabelled with a detectable label, and utilised for diagnostic purposes.Similarly, the thus-labelled polypeptide of the invention may be used toidentify its corresponding receptor in situ. The polypeptide or antibodymay be covalently or non-covalently coupled to a suitable supermagnetic,paramagnetic, electron dense, ecogenic or radioactive agent for imaging.For use in diagnostic assays, radioactive or non-radioactive labels, thelatter including enzyme labels or labels of the biotin/avidin system,may be used.

[0036] Clinical applications of the invention include diagnosticapplications, acceleration of angiogenesis in wound healing, tissue ororgan transplantation, or to establish collateral circulation in tissueinfarction or arterial stenosis, such as coronary artery disease, andinhibition of angiogenesis in the treatment of cancer or of diabeticretinopathy. Quantitation of VEGF-D in cancer biopsy specimens may beuseful as an indicator of future metastatic risk.

[0037] Inasmuch as VEGF-D is highly expressed in the lung, and it alsoincreases vascular permeability, it is relevant to a variety of lungconditions. VEGF-D assays could be used in the diagnosis of various lungdisorders. VEGF-D could also be used in the treatment of lung disordersto improve blood circulation in the lung and/or gaseous exchange betweenthe lungs and the blood stream. Similarly, VEGF-D could be used toimprove blood circulation to the heart and O₂ gas permeability in casesof cardiac insufficiency. In like manner, VEGF-D could be used toimprove blood flow and gaseous exhange in chronic obstructive airwaydisease.

[0038] Conversely, VEGF-D antagonists (e.g. antibodies and/orinhibitors) could be used to treat in conditions, such as congestiveheart failure, involving accumulations of fluid in, for example, thelung resulting from increases in vascular permeability, by exerting anoffsetting effect on vascular permeability in order to counteract thefluid accumulation.

[0039] VEGF-D is also expressed in the small intestine and colon, andadministrations of VEGF-D could be used to treat malabsorptive syndromesin the intestinal tract as a result of its blood circulation increasingand vascular permeabiltiy increasing activities.

[0040] Thus the invention provides a method of stimulation ofangiogenesis and/or neovascularization in a mammal in need of suchtreatment, comprising the step of administering an effective dose ofVEGF-D, or a fragment or analogue thereof which has the ability tostimulate endothelial cell proliferation, to the mammal.

[0041] Optionally VEGF-D may be administered together with, or inconjunction with, one or more of VEGF-A, VEGF-B, VEGF-C, PlGF, PDGF, FGFand/or heparin.

[0042] Conversely the invention provides a method of inhibitingangiogenesis and/or neovascularization in a mammal in need of suchtreatment, comprising the step of administering an effective amount ofan antagonist of VEGF-D to the mammal. The antagonist may be any agentthat prevents the action of VEGF-D, either by preventing the binding ofVEGF-D to its corresponding receptor or the target cell, or bypreventing activation of the transducer of the signal from the receptorto its cellular site of action. Suitable antagonists include, but arenot limited to, antibodies directed against VEGF-D; competitive ornon-competitive inhibitors of binding of VEGF-D to the VEGF-D receptor,such as the receptor-binding but non-mitogenic VEGF-D variants referredto above; and anti-sense nucleotide sequences complementary to at leasta part of the DNA sequence encoding VEGF-D.

[0043] The invention also provides a method of detecting VEGF-D in abiological sample, comprising the step of contacting the sample with areagent capable of binding VEGF-D, and detecting the binding. Preferablythe reagent capable of binding VEGF-D is an antibody directed againstVEGF-D, more preferably a monoclonal antibody. In a preferred embodimentthe binding and/or extent of binding is detected by means of adetectable label; suitable labels are discussed above.

[0044] Where VEGF-D or an antagonist is to be used for therapeuticpurposes, the dose and route of application will depend upon thecondition to be treated, and will be at the discretion of the attendingphysician or veterinarian. Suitable routes include subcutaneous,intramuscular or intravenous injection, topical application, implantsetc. Topical application of VEGF-D may be used in a manner analogous toVEGF.

[0045] According to yet a further aspect, the invention providesdiagnostic/prognostic means typically in the form of test kits. Forexample, in one embodiment of the invention there is provided adiagnostic/prognostic test kit comprising an antibody to VEGF-D andmeans for detecting, and more preferably evaluating, binding between theantibody and VEGF-D. In one preferred embodiment of thediagnostic/prognostic means according to the invention, either theantibody or the VEGF-D is labelled with a detectable label, and eitherthe antibody or the VEGF-D is substrate-bound, such that theVEGF-D-antibody interaction can be established by determining the amountof label attached to the substrate following binding between theantibody and the VEGF-D. In a particularly preferred embodiment of theinvention, the diagnostic/prognostic means may be provided as aconventional ELISA kit.

[0046] In another alternative embodiment, the diagnostic/prognosticmeans may comprise polymerase chain reaction means for establishing thegenomic sequence structure of a VEGF-D gene of a test individual andcomparing this sequence structure with that disclosed in thisapplication in order to detect any abnormalities, with a view toestablishing whether any aberrations in VEGF-D expression are related toa given disease condition.

[0047] In accordance with a further aspect, the invention relates to amethod of detecting aberrations in VEGF-D gene structure in a testsubject which may be associated with a disease condition in said testsubject. This method comprises providing a DNA sample from said testsubject; contacting the DNA sample with a set of primers specific toVEGF-D DNA operatively coupled to a polymerase and selectivelyamplifying VEGF-D DNA from the sample by polymerase chain reaction, andcomparing the nucleotide sequence of the amplified VEGF-D DNA from thesample with the nucleotide sequences set forth in SEQ ID NO:1 or SEQ IDNO:4. The invention also includes the provision of a test kit comprisinga pair of primers specific to VEGF-D DNA operatively coupled to apolymerase, whereby said polymerase is enabled to selectively amplifyVEGF-D DNA from a DNA sample.

[0048] Another aspect of the invention concerns the provision of apharmaceutical composition comprising either VEGF-D polypeptide or afragment or analogue thereof which promotes proliferation of endothelialcells, or an antibody thereto. Compositions which comprise VEGF-Dpolypeptide may optionally further comprise one or more of VEGF, VEGF-Band VEGF-C, and/or heparin.

[0049] In another aspect, the invention relates to a protein dimercomprising VEGF-D polypeptide, particularly a disulphide-linked dimer.The protein dimers of the invention include both homodimers of VEGF-Dpolypeptide and heterodimers of VEGF-D and VEGF, VEGF-B, VEGF-C, PlGF orPDGF.

[0050] According to a yet further aspect of the invention there isprovided a method for isolation of VEGF-D comprising the step ofexposing a cell which expresses VEGF-D to heparin to facilitate releaseof VEGF-D from the cell, and purifying the thus-released VEGF-D.

[0051] Another aspect of the invention involves providing a vectorcomprising an anti-sense nucleotide sequence which is complementary toat least a part of a DNA sequence which encodes VEGF-D or a fragment oranalogue thereof which promotes proliferation of endothelial cells.According to a yet further aspect of the invention such a vectorcomprising an anti-sense sequence may be used to inhibit, or at leastmitigate, VEGF-D expression. The use of a vector of this type to inhibitVEGF-D expression is favoured in instances where VEGF-D expression isassociated with a disease, for example where tumours produce VEGF-D inorder to provide for angiogenesis. Transformation of such tumour cellswith a vector containing an anti-sense nucleotide sequence wouldsuppress or retard angiogenesis, and so would inhibit or retard growthof the tumour.

[0052] Polynucleotides of the invention such as those described above,fragments of those polynucleotides, and variants of thosepolynucleotides with sufficient similarity to the non-coding strand ofthose polynucleotides to hybridise thereto under stringent conditionsall are useful for identifying, purifying, and isolating polynucleotidesencoding other, non-human, mammalian forms of VEGF-D. Thus, suchpolynucleotide fragments and variants are intended as aspects of theinvention. Exemplary stringent hybridisation conditions are as follows:hybridisation at 42° C. in 5×SSC, 20 mM NaPO₄, pH 6.8, 50% formamide;and washing at 42° C. in 0.2×SSC. Those skilled in the art understandthat it is desirable to vary these conditions empirically based on thelength and the GC nucleotide base content of the sequences to behybridised, and that formulae for determining such variation exist. Seefor example Sambrook et al, “Molecular Cloning: A Laboratory Manual”,Second Edition, Cold Spring Harbor, New York: Cold Spring HarborLaboratory (1989).

[0053] Moreover, purified and isolated polynucleotides encoding other,non-human, mammalian VEGF-D forms also are aspects of the invention, asare the polypeptides encoded thereby, and antibodies that arespecifically immunoreactive with the non-human VEGF-D variants. Thus,the invention includes a purified and isolated mammalian VEGF-Dpolypeptide, and also a purified and isolated polynucleotide encodingsuch a polypeptide.

[0054] It will be clearly understood that nucleic acids and polypeptidesof the invention may be prepared by synthetic means or by recombinantmeans, or may be purified from natural sources.

BRIEF DESCRIPTION OF THE FIGURES

[0055]FIG. 1 shows a comparison between the sequences of human VEGF-Dand human VEGF₁₆₅ (FIG. 1a), human VEGF-B (FIG. 1b), human VEGF-C (FIG.1c) and human PlGF (FIG. 1d). The box indicates residues which matchthose in human VEGF-D exactly;

[0056]FIG. 2 shows sequence alignments between the sequences of humanVEGF-D, human VEGF₁₆₅, human VEGF-B, human VEGF-C and human PlGF. Theboxes indicate residues that match the VEGF-B sequence exactly; and

[0057]FIG. 3 shows the amino acid sequence of human VEGF-D (SEQ ID NO3), as predicted from the cDNA sequence (SEQ ID NO 1). The boxesindicate potential sites for N-linked glycosylation.

[0058]FIG. 4 shows the nucleotide sequence of a second cDNA sequenceencoding human VEGF-D (SEQ ID NO 4), isolated by hybridisation from acommercial human lung cDNA library; this cDNA contains the entire codingregion for human VEGF-D;

[0059]FIG. 5 shows the amino acid sequence for human VEGF-D (SEQ ID NO5) deduced from the sequence of the cDNA of FIG. 4;

[0060]FIG. 6 shows the nucleotide sequence of cDNA encoding mouseVEGF-D1 (SEQ ID NO 6), isolated by hybridisation screening for acommercially-available mouse lung cDNA library;

[0061]FIG. 7 shows the nucleotide sequence of cDNA encoding mouseVEGF-D2 (SEQ ID NO 7), isolated from the same library as in FIG. 6;

[0062]FIG. 8 shows the deduced amino acid sequences for mouse VEGF-D1(SEQ ID NO 8) and VEGF-D2 (SEQ ID NO 9);

[0063]FIG. 9 shows a comparison between the deduced amino acid sequencesof mouse VEGF-D1, mouse VEGF-D2 and human VEGF-D;

[0064]FIG. 10 shows sequence alignments between the amino acid sequencesof human VEGF-D, human VEGF₁₆₅, human VEGF-B, human VEGF-C-and humanPlGF; and

[0065]FIG. 11 shows the results of a bioassay in which conditionedmedium from COS cells expressing either VEGF-A or VEGF-D was tested forability to bind to the extracellular domain of a chimeric receptorexpressed in Ba/F3 cells.

[0066]FIG. 12 shows the results of immunoprecipitation and Westernblotting analysis of VEGF-D peptides

[0067] (A) pEFBOSVEGFDfullFLAG and pCDNA-1VEGF-A were transfected intoCOS cells and biosynthetically labelled with ³⁵S-cysteine/methionine for4 hours. The supernatants from these cultures were immunoprecipitatedwith either M2 gel or an antiserum directed to VEGF-A coupled to proteinA. Washed beads were eluted with an equal volume of 2×SDS-PAGE samplebuffer and boiled. The samples were then resolved by 12% SDS-PAGE. Lanesmarked with an asterix (*) indicate where samples were reduced withdithiothreitol and alkylated with iodoacetamide. Molecular weightmarkers are indicated. fA and fB indicate the 43 kD and 25 kD speciesimmunoprecipitated by the M2 gel from the COS cells expressingpEFBOSVEGFDfullFLAG.

[0068] (B) Western blotting analysis of purified VEGFDΔNΔC. An aliquotof material eluted from the M2 affinity column (fraction #3, VEGFDΔNΔC)was combined with 2×SDS-PAGE sample buffer and resolved on a 15%SDS-PAGE gel. The proteins were then transferred to nitrocellulosemembrane and probed with either monoclonal antibody M2 or a controlisotype-matched antibody (Neg). Blots were developed using a goatanti-mouse-HRP secondary antibody and chemiluminescence (ECL, Amersham).Monomeric VEGFDΔNΔC is arrowed, as is the putative dimeric form of thispeptide (VEGFDΔNΔC″). Molecular weight markers are indicated.

[0069]FIG. 13 shows the results of analysis of VEGFDΔNΔC protein usingthe VEGFR2 bioassay. Recombinant VEGFDΔNΔC, and material purified by M2affinity chromatography, was assessed using the VEGFR2 bioassay.Bioassay cells (10⁴), washed to remove IL-3, were incubated withaliquots of conditioned medium from VEGF-D transfected COS cells,fraction #1 from the affinity column (void volume) or fraction #3 fromthe affinity column (containing VEGFDΔNΔC). All samples were tested atan initial concentration of 20% (ie ⅕) followed by doubling dilutions.Cells were allowed to incubate for 48 hours at 37° C. in a humidifiedatmosphere of 10% CO₂. Cell proliferation was quantitated by theaddition of 1 μCi of ³H-thymidine and counting the amount incorporatedover a period of 4 hours.

[0070]FIG. 14 shows stimulation of tyrosine phosphorylation of theVEGFR3 receptor (Flt4) on NIH3T3 cells by culture supernatant from HFcells infected with a recombinant baculovirus vector transformed withVEGF-D.

[0071]FIG. 15 shows stimulation of tyrosine phosphorylation of theVEGFR2 receptor (KDR) in PAE cells by culture supernatant prepared as inFIG. 14.

[0072]FIG. 16 shows the mitogenic effect of VEGFDΔNΔC on bovine aorticendothelial cells (BAEs). BAEs were treated with fraction #3 containingVEGFDΔNΔC and, as positive control, purified VEGF-A as described in thetext. The result obtained using medium without added growth factor isdenoted Medium Control.

DETAILED DESCRIPTION OF THE INVENTION

[0073] The invention will now be described in detail by reference to thefigures, and to the following non-limiting examples.

EXAMPLE 1

[0074] It has been speculated that no further members of the VEGF familywill be found, because there are no known orphan receptors in the VEGFRfamily. Furthermore, we are not aware of any suggestion in the prior artthat other such family members would exist.

[0075] A computer search of nucleic acid databases was carried outincidentally to another project, using as search topics the amino acidsequences of VEGF, VEGF-B, VEGF-C and PlGF. Several cDNA sequences wereidentified by this search. One of these sequences, GenBank Accession No.H24828, encoded a polypeptide which was similar in structure to thecysteine-riched C-terminal region of VEGF-C. This sequence was obtainedfrom the database of expressed sequence tags (dbEST), and for thepurposes of this specification is designated XPT. The XPT cDNA had beenisolated from a human cDNA library designated “Soares Breast 3NbHBst”,which was constructed using mRNA from an adult human female breasttissue. As far as can be ascertained this was normal breast tissue.Sequencing of the XPT DNA was performed pursuant to the IntegratedMolecular Analysis of Genome Expression Consortium (IMAGE Consortium),which solicits cDNA libraries from laboratories around the world, arraysthe cDNA clones, and provides them to other organisations forsequencing.

[0076] The XPT sequence shown in the database was 419 nucleotides long,and encoded an amino acid sequence similar to the C-terminal 100 aminoacids of VEGF-C, ie. approximately residues 250 to 350, using thenumbering system of Joukov et al (1996). Similarly cysteine-rich regionsare found in other proteins, which are entirely unrelated in function tothe VEGF family, for example the secreted silk-like protein sp185synthesized in the salivary glands of the midge Chironomus tentans. Thisprotein is encoded by the gene BR3, located in a Balbiani ring, a tissuespecific chromosome “puff” found on polytene chromosomes in the midgesalivary gland (Dignam and Case: Gene, 1990 88 133-140; Paulsson et al,J. Mol. Biol., 1990 211 331-349). It is stated in Joukov et al (1996)that the sp185-like structural motif in VEGF-C may fold into anindependent domain, which is thought to be at least partially cleavedoff after biosynthesis, and that there is at least one cysteine motif ofthe sp185 type in the C-terminal region of VEGF.

[0077]FIG. 3 of Joukov et al shows that the last two-thirds of theC-terminal cysteine-rich region of VEGF-C do not align with VEGF orPlGF, and in fact could be considered a C-terminal extension of VEGF-Cwhich is not present in VEGF or PlGF. The sequence encoded by XPT issimilar to this extension. As the XPT cDNA was truncated at its 5′ end,it was not possible to deduce or predict any amino acid sequence forregions N-terminal to the cysteine-rich domain. Thus the portion ofVEGF-C which is similar to the XPT-derived sequence does not extend toregions of VEGF-C which are conserved among other members of the VEGFfamily.

[0078] As described above, it was not possible to predict whether theN-terminal region of the polypeptide encoded by a full-length XPTnucleic acid (as distinct from the truncated XPT cDNA reported in dbEST)would show any further homology to any member of the VEGF family, inparticular VEGF-C, which has a further N-terminal 250 amino acids. Forexample, the naturally-occurring protein encoded by a full-length XPTnucleic acid could have been the human homologue of the midge salivarygland protein. Alternatively, the type of cysteine-rich motif encoded bytruncated XPT cDNA could be widely distributed among proteins, as aremany structural domains. For example, clusters of cysteine residues maybe involved in metal binding, formation of intramolecular disulphidebonds to promote accurate protein folding, or formation ofintermolecular disulphide bonds for assembly of protein subunits intocomplexes (Dignam and Chase, 1990). In order to determine whether thetruncated XPT cDNA was derived from sequences encoding a VEGF-relatedmolecule, it was necessary to isolate a much longer cDNA.

EXAMPLE 2 Cloning of cDNA Encoding VEGF-D

[0079] A sample of the XPT cDNA reported in dbEST was obtained from theAmerican Type Culture Collection, which is a registered supplier of cDNAclones obtained by the IMAGE Consortium. The identity of the XPT cDNAwas confirmed by nucleotide sequencing, using the dideoxy chaintermination method (Sanger et al, Proc. Natl. Acad. Sci. USA, 1977 745463-5467).

[0080] The XPT cDNA was used as a hybridization probe to screen a humanbreast cDNA library, which was obtained commercially from Clontech. Onepositive clone was isolated, and this clone was then sequenced on bothstrands. The nucleotide sequence was compiled, and an open reading framewas identified. The nucleic acid sequence is set out in SEQ ID NO. 1.The polypeptide encoded by this sequence was designated VEGF-D, and itsdeduced amino acid sequence, designated SEQ ID NO. 3, is set out in FIG.3. In FIG. 3 putative sites of N-linked glycosylation, with theconsensus sequence N-X-S/T in which X is any amino acid, are indicatedby the boxes.

EXAMPLE 3 Characteristics of VEGF-D

[0081] The amino acid sequence of VEGF-D was compared with those ofhuman VEGF-A₁₆₅, VEGF-B, VEGF-C and PlGF. These comparisons are set outin FIGS. 1a to d respectively. The degree of sequence homology wascalculated, and if gaps in sequence introduced for the purposes ofalignment are not considered in the calculation, VEGF-D is 31% identicalto VEGF, 48% identical to VEGF-C, 28% identical to VEGF-B and 32%identical to PlGF. Thus the most closely-related protein identified wasVEGF-C.

[0082] Computer searches of the GenBank, EMBL and SwissProt nucleic aciddatabases did not reveal any protein sequences identical to VEGF-D. Asexpected from the sequence alignment referred to above, the most closelyrelated protein found in these databases was VEGF-C. Searches of dbESTwere also performed, but did not reveal any sequences encompassing theentire coding region of VEGF-D. The sequence of VEGF-D is unrelated tothat of Tie-2 ligand 1 as disclosed in WO 96/11269.

[0083] It is important to bear in mind that the only homologies detectedwere at the level of amino acid sequence. Thus it would not have beenpossible to isolate the cDNA or gDNA encoding VEGF-D by methods such aslow-stringency hybridization with a nucleic acid sequence encodinganother member of the VEGF family.

[0084] VEGF-D appears to be most closely related to VEGF-C of all themembers of the VEGF family. Because the VEGF-D amino acid sequenceincludes the cysteine-rich sp185-like motif which is found in VEGF-C,the polypeptide of the invention may play an important functional rolein lymphatic endothelia. While we do not wish to be bound by anyproposed mechanism, it is thought that VEGF-C and VEGF-D may constitutea silk-like matrix over which endothelial cells can grow. Lymphaticvessels have no basement membrane, so the silk-like matrix can form abasement membrane-like material. This may be important in promoting cellgrowth and/or in cell differentiation, and may be relevant to cancer,especially metastasis, drug therapy, cancer prognosis, etc.

EXAMPLE 4 Biological Characteristics of VEGF-D

[0085] The cDNA sequence of VEGF-D was used to predict the deduced aminoacid sequence of VEGF-D, the biochemical characteristics of the encodedpolypeptide, including the numbers of strongly basic, strongly acidic,hydrophobic and polar amino acids, the molecular weight, the isoelectricpoint, the charge at pH 7, and the compositional analysis of the wholeprotein. This analysis was performed using the Protean protein analysisprogram, Version 1.20 (DATASTAR). These results are summarised in Tables1 and 2 below. Table 1 also shows the codon usage. TABLE 1 TranslatedDNA Sequence of VEGF-D contig × (1,978) With Standard Genetic CodeMolecular Weight 37056.60 Daltons 425 Amino Acids 46 Strong Basic (+)Amino Acids (K,R) 41 Strong Acidic (−) Amino Acids (D,E) 79 hydrophobicAmino Acids (A,I,L,F,W,V) 108 Polar Amino Acids (N,C,Q,S,T,Y) 7.792Isoelectric Point 6.371 Charge at pH 7.0 Total number of basestranslated is 978 % A = 28.73 [281] % G = 23.11 [226] % T = 23.21 [227]% C = 24.95 [244] % Ambiguous = 0.00 [0] % A + T = 51.94 [508] % C + G =48.06 [470] Davis, Botstein, Roth Melting Temp ° C. 84.09 Wallace Temp °C. 3384.00 Codon usage: ccg ( )  0 # ugc Cys (C) 14 # cuc Leu (L)  6 #ucg Ser (S) uaa ( )  0 # ugu Cys (C) 16 # cug Leu (L)  4 # ucu Ser (S)uag ( )  0 # ——— Cys (C) 30 # cuu Leu (L)  2 # ——— Ser (S) 3 ——— ( )  0# caa Gln (Q)  1 # uua Leu (L)  1 # uga Ter (.) gca Ala (A)  5 # cag Gln(Q) 11 # uug Leu (L)  5 # ——— Ter (.) gcc Ala (A)  4 # ——— Gln (Q) 12 #——— Leu (L) 23 # aca Thr (T) gcg Ala (A)  1 # gaa Glu (E) 16 # aaa Lys(K) 13 # acc Thr (T) gcu Ala (A)  5 # gag Glu (E) 12 # aag Lys (K) 10 #acg Thr (T) ——— Ala (A) 15 # ——— Glu (E) 28 # ——— Lys (K) 23 # acu Thr(T) aga Arg (R)  7 # gga Gly (G)  1 # aug Met (M)  6 # ——— Thr (T) 2 aggArg (R)  5 # ggc Gly (G)  2 # ——— Met (M)  6 # ugg Trp (W) cga Arg (R) 5 # ggg Gly (G)  3 # uuc Phe (F)  4 # ——— Trp (W) cgc Arg (R)  4 # gguGly (G)  2 # uuu Phe (F)  8 # uac Tyr (Y) cgg Arg (R)  1 # ——— Gly (G) 8 # ——— Phe (F) 12 # uau Tyr (Y) cgu Arg (R)  1 # cac His (H)  7 # ccaPro (P)  9 # ——— Tyr (Y) ——— Arg (R) 23 # cau His (H)  7 # ccc Pro (P) 6 # gua Val (V) aac Asn (N)  5 # ——— His (H) 14 # ccu Pro (P)  8 # gucVal (V) aau Asn (N)  4 # aua Ile (I)  2 # ——— Pro (P) 23 # gug Val (V)——— Asn (N)  9 # auc Ile (I)  6 # agc Ser (S)  6 # guu Val (V) gac Asp(D)  8 # auu Ile (I)  5 # agu Ser (S)  8 # ——— Val (V) 1 qau Asp (D)  5# ——— Ile (I) 13 # uca Ser (S)  5 # nnn ??? (X) gau Asp (D)  5 # ——— Ile(I) 13 # uca Ser (S)  5 # nnn ??? (X) ——— Asp (D) 13 # cua Leu (L)  5 #ucc Ser (S)  7 # TOTAL 32

[0086] TABLE 2 Predicted Structural Class of the Whole Protein: Deléage& Roux Modification of Nishikawa & Ooi 1987 Analysis Whole ProteinMolecular Weight 37056.60 m.w. Length 325 1 microgram = 26.986 pMolesMolar Extinction 30200 ± 5% coefficient 1 A(280) = 1.23 mg/mlIsoelectric Point 7.79 Charge at pH 7 6.37 Whole Protein CompositionAnayalsis Amino Acid(s) Number count % by weight % by frequency Charged(RKHYCDE) 134 46.30 41.23 Acidic (DE) 41 13.79 12.62 Basic (KR) 46 17.6514.15 Polar (NCQSTY) 108 30.08 33.23 Hydrophobic (AILFWV) 79 23.86 24.31A Ala 15 2.88 4.62 C Cys 30 8.35 9.23 D Asp 13 4.04 4.00 E Glu 28 9.758.62 F Phe 12 4.77 3.69 G Gly 8 1.23 2.46 H His 14 5.18 4.31 I Ile 133.97 4.00 K Lys 23 7.96 7.08 L Leu 23 7.03 7.08 M Met 6 2.12 1.85 N Asn9 2.77 2.77 P Pro 23 6.08 7.08 Q Gln 12 4.15 3.69 R Arg 23 9.69 7.08 SSer 33 7.76 10.15 T Thr 21 5.73 6.46 V Val 12 3.21 3.69 W Trp 4 2.011.23 Y Trp 3 1.32 0.92 B Asx 0 0.00 0.00 Z Glx 0 0.00 0.00 X Xxx 0 0.000.00 . Ter 0 0.00 0.00

[0087] This analysis predicts a molecular weight for the unprocessedVEGF-D monomer of 37 kilodaltons (kD), compared to the experimentallydetermined values (for the fully processes peptides) of 20 to 27 kD forVEGF-A monomers, 21 kD for the VEGF-B monomer and 23 kD for the VEGF-Cmonomer.

EXAMPLE 5

[0088] The original isolation of a cDNA for VEGF-D, described in Example2 involved hybridisation screening of a human breast cDNA library. Asonly one cDNA clone for VEGF-D was thus isolated, it was not possible toconfirm the structure of the cDNA by comparison with other independentlyisolated VEGF-D cDNAs. The work described in this example, whichinvolved isolation of additional human VEGF-D cDNA clones, was carriedout in order to confirm the structure of human VEGF-D cDNA. In addition,mouse VEGF-D cDNA clones were isolated.

[0089] Two cDNA libraries which had been obtained commercially fromStratagene, one for human lung and one for mouse lung (catalogue numbers937210 and 936307, respectively) were used for hybridisation screeningwith a VEGF-D cDNA probe. The probe, which spanned from nucleotides 1817to 2495 of the cDNA for human VEGF-D described in Example 2, wasgenerated by polymerase chain reaction (PCR) using a plasmid containingthe VEGF-D cDNA as template and the following two oligonucleotides:

[0090] 5′-GGGCTGCTTCTAGTTTGGAG (SEQ ID NO. 10), and

[0091] 5′-CACTCGCAACGATCTTCGTC (SEQ ID NO. 11).

[0092] Approximately two million recombinant bacteriophage were screenedwith this probe from each of the two cDNA libraries. Nine human and sixmouse cDNA clones for VEGF-D were subsequently isolated.

[0093] Two of the nine human cDNA clones for VEGF-D were sequencedcompletely using the dideoxy chain termination method (Sanger et al,Proc. Natl. Acad. Sci. USA, 1977 74 5463-5467). The two cDNAs containedthe entire coding region for human VEGF-D, and were identical exceptthat one of the clones was five nucleotides longer than the other at the5′-terminus. The nucleotide sequence of the shorter cDNA is shown inFIG. 4, and is designated SEQ ID NO. 4. The amino acid sequence forhuman VEGF-D (hVEGF-D) deduced from this cDNA was 354 residues long, andis shown in FIG. 5; this is designated SEQ ID NO. 5. The sequences ofthe 5′ regions of five of the other human VEGF-D cDNA clones were alsodetermined. For each clone, the sequence that was characterizedcontained more than 100 nucleotides of DNA immediately downstream fromthe translation start site of the coding region. In all cases, thesequences of these regions were identical to corresponding regions ofthe human VEGF-D cDNA shown in FIG. 4.

[0094] All six mouse cDNA clones for VEGF-D were sequenced completely.Only two of the clones contained an entire coding region for VEGF-D; theother clones were truncated. The nucleotide sequences of the two cloneswith an entire coding region are different, and encode amino acidsequences of different sizes. The longer amino acid sequence isdesignated mVEGF-D1, and the shorter sequence is designated mVEGF-D2.The nucleotide sequences of the cDNAs encoding mVEGF-D1 and mVEGF-D2 areshown in FIGS. 6 and 7 respectively. The deduced amino acid sequencesfor mVEGF-D1 and mVEGF-D2 are shown in FIG. 8. These sequences arerespectively designated SEQ ID NOS. 6, 7, 8 and 9. The differencesbetween the amino acid sequences are:

[0095] i) an insertion of five amino acids (DFSFE) after residue 30 inmVEGF-D1 in comparison to mVEGF-D2;

[0096] ii) complete divergence of the C-terminal ends after residue 317in mVEGF-D1 and residue 312 in mVEGF-D2, which results in mVEGF-D1 beingconsiderably longer.

[0097] Three of the four truncated cDNAs for mouse VEGF-D encoded theC-terminal region, but not the N-terminal 50 amino acids. All three ofthese cDNAs encoded a C-terminal end for VEGF-D which is identical tothat for mVEGF-D2. The other truncated cDNA encoded only the N-terminalhalf of VEGF-D. The amino acid sequence deduced from this cDNA containedthe five amino acids DFSFE immediately after residue 30 found inmVEGF-D1, but not in mVEGF-D2.

[0098] As described above, the entire sequence of the human VEGF-D cDNAclone reported in this example has been validated by comparison withthat for a second human clone. In addition, the sequence of the 5′ endof the coding region was found to be identical in five other humanVEGF-D cDNA clones. In contrast, the sequence reported in Example 2contained most of the coding region for VEGF-D, but was incorrect nearthe 5′-end of this region. This was probably because the VEGF-D cDNA wastruncated near the 5′-end of the coding region and at that point hadbeen ligated with another unidentified cDNA, and consequently the first30 codons of the true coding sequence for VEGF-D had been deleted andreplaced with a methionine residue. This methionine residue was definedas the N-terminal amino acid of the VEGF-D sequence presented in Example2.

[0099] The N-terminal regions of the deduced amino acid sequences ofmouse VEGF-D1 and VEGF-D2 are very similar to that deduced for humanVEGF-D (see FIG. 9). This also indicates that the correct deduced aminoacid sequence for human VEGF-D is reported in this example. TheN-terminal 25 amino acids of human VEGF-D form an extremely hydrophobicregion, which is consistent with the notion that part of this region maybe a signal sequence for protein secretion. FIG. 10 shows the alignmentof the human VEGF-D sequence with the sequences of other members of theVEGF family of growth factors, namely human VEGF₁₆₅ (hVEGF₁₆₅), humanVEGF-B (hVEGF-B), human VEGF-C (hVEGF-C) and human Placental GrowthFactor (hPlGF). When gaps in the alignments are ignored for the purposesof calculation, human VEGF-D is found to be 31% identical in amino acidsequence to human VEGF₁₆₅, 28% identical to human VEGF-B, 48% identicalto VEGF-C and 32% identical to human PlGF. Clearly VEGF-C is the memberof this family which is most closely related to VEGF-D.

[0100] The differences in sequence for mouse VEGF-D1 and VEGF-D2 mostprobably arise from differential mRNA splicing. The C-terminal 41 aminoacid residues of VEGF-D1 are deleted in VEGF-D2, and are replaced with 9residues which are not closely related to the VEGF-D1 sequence.Therefore 4 cysteine residues present near the C-terminus of VEGF-D1 aredeleted in VEGF-D2. This change may alter the tertiary or quaternarystructures of the protein, or may affect the localisation of the proteinin the cell or the extracellular environment. The C-terminal end ofhuman VEGF-D resembles that of mouse VEGF-D1, not mouse VEGF-D2. Thesmall 5 amino acid insertion after residue 30 in mouse VEGF-D1, which isnot present in either mouse VEGF-D2 or human VEGF-D, may influenceproteolytic processing of the protein.

[0101] VEGF-D is highly conserved between mouse and man. Eighty-fivepercent of the amino acid residues of human VEGF-D are identical inmouse VEGF-D1. This is likely to reflect conservation of proteinfunction. Putative functions for VEGF-D have been proposed herein.Although we have not found alternative forms of human VEGF-D cDNA, it ispossible that the RNA splice variation which gives rise to numerousforms of mRNA for mouse VEGF-D may also occur in human tissues.

EXAMPLE 6 Expression of VEGF-D in COS Cells

[0102] A fragment of the human cDNA for VEGF-D, spanning from nucleotide1 to 1520 of the sequence shown in FIG. 4 and containing the entirecoding region, was inserted into the mammalian expression vectorpCDNA1-amp. The vector was used to transiently transfect COS cells bythe DEAE-Dextran method as described previously (Aruffo and Seed, 1987)and the resulting conditioned cell culture media, collected after 7 daysof incubation, were concentrated using Amicon concentrators (Centricon10 with a 10,000 molecular weight cut off) according to themanufacturer. The plasmids used for transfections were the expressionconstruct for human VEGF-D and, as positive control, a construct made byinsertion of mouse VEGF-A cDNA into pCDNA1-amp. The conditioned mediawere tested in two different bioassays, as described below, and theresults demonstrate that the COS cells did in fact express and secretebiologically-active VEGF-D.

EXAMPLE 7 Bioassay for Capacity of VEGF-D to Bind to VEGF Receptor-2

[0103] As shown in Example 5, VEGF-D is closely related in primarystructure to other members of the VEGF family. Most members of thisprotein family are mitogenic and/or chemotactic for endothelial cells(Keck et al, 1989; Leung et al, 1989; Joukov, et al, 1996; Olofsson etal, 1996) In addition VEGF-A (previously known as VEGF), the firstmember of the VEGF family to be described in the literature, is a potentinducer of vascular permeability (Keck et al, 1989). As proteinstructure is an important determinant of protein function, it seemedlikely that VEGF-D might also be mitogenic for endothelial cells orinduce vascular permeability. Therefore human VEGF-D was tested in abioassay for its capacity to bind to VEGF receptor-2 (VEGFR2; also knownas Flk-1), an endothelial cell-specific receptor which, when activatedby VEGF-A, is thought to give rise to a mitogenic signal (Strawn et al,1996).

[0104] A bioassay for detection of growth factors which bind to VEGFR2has been developed in the factor-dependent cell line Ba/F3, and isdescribed in our earlier patent application, No. PCT/US95/16755. Thesecells grow in the presence of interleukin-3 (IL-3); however removal ofthis factor results in cell death within 48 hours. If another receptorcapable of delivering a growth stimulus is transfected into the Ba/F3cells, the cells can be rescued by the specific growth factor whichactivates that receptor when the cells are grown in medium lacking IL-3.In the specific case of receptor-type tyrosine kinases (eg. VEGFR2),chimeric receptors containing the extracellular domain of the receptortyrosine kinase and the transmembrane and cytoplasmic domains of theerythropoietin receptor (EpoR) can be utilised. In this case stimulationwith the ligand (eg. VEGF), which binds to the extracellular domain ofthe chimeric receptor, results in signalling via the EpoR cytoplasmicdomain and subsequent rescue of the cell line in growth medium lackingIL-3. The construction of the chimeric receptor used in this study,consisting of the mouse VEGFR2 extracellular domain and the mouse EpoRtransmembrane and cytoplasmic domains, and the bioassay itself aredescribed below.

[0105] Plasmid Construction

[0106] i) Construction of a Plasmid for Generating Chimeric VEGFR2Receptors

[0107] To obtain a plasmid construct with which DNA encoding theextracellular domain of mouse VEGFR2 could easily be ligated with DNAencoding other protein domains, site-directed mutagenesis was usedto-generate a BglII restriction enzyme site at the position of mouseVEGFR2 cDNA which encoded the junction of the extracellular domain andthe transmembrane domain. The full-length clone of the mouse VEGFR2 cDNAdescribed by Oelrichs et al (1993) was subcloned into the mammalianexpression vector pCDNA1-amp, using the BstXI restriction enzyme site.Single stranded UTP+ DNA was generated using the M13 origin ofreplication, and this was used as a template to generate mouse VEGFR2cDNA containing the BglII site at the desired position. The plasmidcontaining the altered VEGFR2 cDNA was designated pVEGFR2Bgl. DNAfragments encoding the transmembrane and cytoplasmic domains of anyreceptor can be inserted at the BglII site of pVEGFR2Bgl in order togenerate chimeric VEGFR2 receptors.

[0108] ii) Construction of VEGFR2/EpoR Chimeric Receptor

[0109] The mouse EpoR cDNA was subcloned into the expression vectorpCDNA1-amp, and single stranded DNA was generated as a template formutagenesis. A BglII restriction enzyme site was inserted into the EpoRcDNA at the position encoding the junction of the transmembrane andextracellular domains of the EpoR to allow direct ligation of this DNAfragment to the modified cDNA encoding the extracellular domain ofVEGFR2 in pVEGFR2Bgl. In addition a BglII site in the cytoplasmic domainof the EpoR was removed by a silent single nucleotide substitution. TheDNA fragment encoding the transmembrane and cytoplasmic domains of EpoRwas then used to replace the portion of pVEGFR2Bgl encoding thetransmembrane and cytoplasmic domains of VEGFR2. Thus a single readingframe was generated which encoded the chimeric receptor consisting ofthe VEGFR2 extracellular domain and the EpoR transmembrane andcytoplasmic domains.

[0110] The DNA fragment encoding the chimeric receptor was subclonedinto the expression vector pBOS, and co-transfected into the Ba/F3 cellline with plasmid pgk-neo at a ratio of 1:20. Cells expressing theVEGFR2-EpoR protein were selected by flow cytometry analysis using amonoclonal antibody to the VEGFR2 extracellular domain (MAb 4H3). Thismonoclonal antibody is described in Australian Patent Application No. PM3794 filed Feb. 10, 1994. Cell lines expressing higher levels ofVEGFR2-EpoR were selected by growing the cells in 5 μg/ml MAb 4H3 or 25ng/ml of recombinant VEGF. A cell line expressing high levels ofVEGFR2-EpoR, designated Ba/F3-NYK-EpoR, was used for the bioassay.

[0111] The Bioassay

[0112] The Ba/F3-NYK-EpoR cells described above were washed three timesin PBS to remove all IL-3 and resuspended at a concentration of 1000cells per 13.5 μl of culture medium and 13.5 μl was aliquoted per wellof a 60-well Terasaki plate. Conditioned media from transfected COScells were then diluted into the cell culture medium. Cells expressing achimeric receptor consisting of the extracellular domain of theendothelial cell receptor Tie2 and the transmembrane and cytoplasmicdomains of EpoR were used as a non-responding control cell line. Cellswere incubated for 48-96 hours, during which the cells incubated in cellculture medium alone had died and the relative survival/proliferationseen in the other wells (ie. in the presence of COS cell-conditionedmedia) was scored by counting the viable cells present per well.

[0113] The conditioned medium from COS cells which had been transientlytransfected with expression plasmids was concentrated 30-fold and usedin the VEGFR2 bioassay. Concentrated conditioned medium from COS cellstransfected with pCDNA1-amp was used as negative control.

[0114] The results are shown in FIG. 11, with the percentage of 30-foldconcentrated COS cell-conditioned medium in the incubation medium(vol/vol) plotted versus the number of viable cells in the well after 48hours of incubation. Clearly the conditioned medium containing eitherVEGF-A or VEGF-D was capable of promoting cell survival in this assay,indicating that both proteins can bind to and activate VEGFR2.

EXAMPLE 8 Vascular Permeability Assay

[0115] Human VEGF-D, prepared as in Example 6 and concentrated 30-fold,was tested in the Miles vascular permeability assay (Miles and Miles,1952) performed in anaesthetized guinea pigs (albino/white, 300-400 g).Concentrated conditioned medium for COS cells transfected withpCDNA1-amp was again used as a negative control. Guinea pigs wereanaesthetised with chloral-hydrate (3.6 g/100 ml; 0.1 ml per 10 g ofbody weight). The backs of the animals were then carefully shaved withclippers. Animals were given an intracardiac injection of Evans Blue dye(0.5% in MT PBS, 0.5 ml) using a 23 G needle, and were then injectedintra-dermally with 100-150 μl of concentrated COS cell-conditionedmedium. After 15-20 min the animals were sacrificed and the layer ofskin on the back excised to expose the underlying blood vessels. Forquantitation, the area of each injection was excised and heated to 45°C. in 2-5 ml of formamide. The resulting supernatants, containingextravasated dye, were then examined spectrophotometrically at 620 nm.

[0116] For animal 1, the absorbance at 620 nm arising from injection of30-fold concentrated VEGF-A conditioned medium was 0.178, that for the30-fold concentrated VEGF-D conditioned medium was 0.114, and that for30-fold concentrated medium from cells transfected with pCDNA1-amp was0.004. For animal 2, the 30-fold concentrated media were diluted 4-foldin cell culture medium before intra-dermal injection. The absorbance at620 nm for the VEGF-A conditioned sample was 0.141, that for the VEGF-Dconditioned sample was 0.116 and that for a sample matched for serumcontent as negative control was 0.017. The enhanced extravasation of dyeobserved for both animals in the presence of VEGF-A or VEGF-Ddemonstrated that both of these proteins strongly induced vascularpermeability.

[0117] The data described here indicate that VEGF-D is a secretedprotein which, like VEGF-A, binds to and activates VEGFR2 and can inducevascular permeability.

EXAMPLE 9 Bioactivities of Internal VEGF-D Polypeptides

[0118] The deduced amino acid sequence for VEGF-D includes a centralregion which is similar in sequence to all other members of the VEGFfamily (approximately residues 101 to 196 of the human VEGF-D amino acidsequence as shown in the alignment in FIG. 10). Therefore, it wasthought that the bioactive portion of VEGF-D might reside in theconserved region. In order to test this hypothesis, the biosynthesis ofVEGF-D was studied, and the conserved region of human VEGF-D wasexpressed in mammalian cells, purified, and tested in bioassays asdescribed below.

[0119] Plasmid Construction

[0120] A DNA fragment encoding the portion of human VEGF-D from residue93 to 201, ie. with N- and C-terminal regions removed, was amplified bypolymerase chain reaction with Pfu DNA polymerase, using as template aplasmid comprising full-length human VEGF-D cDNA. The amplified DNAfragment, the sequence of which was confirmed by nucleotide sequencing,was then inserted into the expression vector pEFBOSSFLAG to give rise toa plasmid designated pEFBOSVEGFDΔNΔC. The pEFBOSSFLAG vector containsDNA encoding the signal sequence for protein secretion from theinterleukin-3 (IL-3) gene and the FLAG™ octapeptide. The FLAG™octapeptide can be recognized by commercially available antibodies suchas the M2 monoclonal antibody (IBI/Kodak). The VEGF-D PCR fragment wasinserted into the vector such that the IL-3 signal sequence wasimmediately upstream from the FLAG™ sequence, which was in turnimmediately upstream from the VEGF-D sequence. All three sequences werein the same reading frame, so that translation of mRNA resulting fromtransfection of pEFBOSVEGFDΔNΔC into mammalian cells would give rise toa protein which would have the IL-3 signal sequence at its N-terminus,followed by the FLAG™ octapeptide and the VEGF-D sequence. Cleavage ofthe signal sequence and subsequent secretion of the protein from thecell would give rise to a VEGF-D polypeptide which is tagged with theFLAG™ octapeptide adjacent to the N-terminus. This protein wasdesignated VEGFDΔNΔC.

[0121] In addition, a second plasmid was constructed, designatedpEFBOSVEGFDfullFLAG, in which the full-length coding sequence of humanVEGF-D was inserted into pEFBOSIFLAG such that the sequence for theFLAG™ octapeptide was immediately downstream from, and in the samereading frame as, the coding sequence of VEGF-D. The plasmid pEFBOSIFLAGlacks the IL-3 signal sequence, so secretion of the VEGF-D/FLAG fusionprotein was driven by the signal sequence of VEGF-D. pEFBOSVEGFDfullFLAGwas designed to drive expression in mammalian cells of full-lengthVEGF-D which was C-terminally tagged with the FLAG™ octapeptide. Thisprotein is designated VEGFDfullFLAG, and is useful for the study ofVEGF-D biosynthesis.

[0122] Analysis of the Post-Translational Processing of VEGF-D

[0123] To examine whether the VEGF-D polypeptide is processed to give amature and fully active protein, pEFBOSVEGFDfullFLAG was transientlytransfected into COS cells (Aruffo and Seed, 1987). Expression in COScells followed by biosynthetic labeling with ³⁵S-methionine/cysteine andimmunoprecipitation with M2 gel has demonstrated species ofapproximately 43 kD (fA) and 25 kD(fB) (FIG. 12A). These bands areconsistent with the notion that VEGF-D is cleaved to give a C-terminalfragment (FLAG™ tagged) and an internal peptide (correspondingapproximately to the VEGFDΔNΔC protein). Reduction of theimmunoprecipitates (M2*) gives some reduction of the fA band, indicatingthe potential for disulphide linkage between the two fragments.

[0124] Expression and Purification of Internal VEGF-D Polypeptide

[0125] Plasmid pEFBOSVEGFDΔNΔC was used to transiently transfect COScells by the DEAE-Dextran method as described previously (Aruffo andSeed, 1987). The resulting conditioned cell culture medium(approximately 150 ml), collected after 7 days of incubation, wassubjected to affinity chromatography using a resin to which the M2monoclonal antibody had been coupled. In brief, the medium was runbatch-wise over a 1 ml M2 antibody column for approximately 4 hours at4° C. The column was then washed extensively with 10 mM Tris-HCl, pH8.0, 150 mM NaCl before elution with free FLAG™ peptide at 25 μg/ml inthe same buffer. The resulting material was used for the bioassaysdescribed below.

[0126] In order to detect the purified VEGFDΔNΔC, fractions eluted fromthe M2 affinity column were subjected to Western blot analysis. Aliquotsof the column fractions were combined with 2×SDS-PAGE sample buffer,boiled and loaded onto a 15% SDS polyacrylamide gel. The resolvedfractions were transferred to nitrocellulose membrane and non-specificbinding sites blocked by incubation in Tris/NaCl/Tween 20 (TST) and 10%skim milk powder (BLOTTO). Membranes were then incubated with monoclonalantibody M2 or control antibody at 3 μg/ml for 2 h at room temperature,followed by extensive washing in TST. Membranes were then incubated witha secondary goat anti-mouse HRP-conjugated antiserum for 1 h at roomtemperature, followed by washing in TST buffer. Detection of the proteinspecies was achieved using a chemiluminescent reagent (ECL, Amersham)(FIG. 12B).

[0127] Under non-reducing conditions a species of molecular weightapproximately 23 kD (VEGFDΔNΔC) was detected by the M2 antibody. This isconsistent with the predicted molecular weight for this internalfragment (12,800) plus N-linked glycosylation; VEGFDΔNΔC contains twopotential N-linked glycosylation sites. A species of approximately 40 kDwas also detected, and may represent a non-covalent dimer of the 23 kDprotein (VEGFDΔNΔC).

[0128] Bioassays

[0129] The bioassay for the capacity of polypeptides to bind to VEGFreceptor-2 is described in detail in Example 7. Aliquots of fractionseluted from the M2 affinity column, containing the VEGFDΔNΔC protein,were diluted in medium and tested in the VEGFR2 bioassay as previouslydescribed. Fraction #3 from the affinity column, which was shown tocontain the purified VEGFDΔNΔC protein (FIG. 12B), demonstrated a clearability to induce proliferation of the bioassay cell line to a dilutionof {fraction (1/100)} of the purified fraction (FIG. 13). In comparison,the void volume of the affinity column (fraction #1) showed no activity,whereas the original VEGFDΔNΔC conditioned medium gave only weakactivity.

[0130] The vascular permeability assay (Miles and Miles, 1952) isdescribed in brief in example 8. Aliquots of purified VEGFDΔNΔC, andsamples of the void volume from the M2 affinity column (negativecontrol) were combined with medium and injected intradermally into theskin of guinea pigs. The regions of skin at the sites of injections wereexcised, and extravasated dye was eluted. The absorbance of theextravasated dye at 620 nm arising from injection of purified VEGFDΔNΔCwas 0.131±0.009. In comparison, the value for absorbance arising frominjection of a sample of the void volume was 0.092±0.020. ThereforeVEGFDΔNΔC induced vascular permeability, but the effect was onlymarginal.

[0131] Due to its ability to bind to VEGFR2 and its lower induction ofvascular permeability compared to full length VEGF-D, VEGF-DΔNΔC may besaid to relatively decrease the induction of vascular permeability byVEGF-D through competitive inhibition. In this sense, the VEGF-DΔNΔCfragment may be thought of as an antagonist for VEGF-D as regards theinduction of vascular permeability.

[0132] Summary

[0133] Two factors have led us to explore internal fragments of VEGF-Dfor enhanced activity. Firstly, it is the central region of VEGF-D whichexhibits amino acid homology with all other members of the VEGF family.Secondly, proteolytic processing which gives rise to internal bioactivepolypeptides occurs for other growth factors such as PDGF-BB. Inaddition, the activity seen with the full length VEGF-D protein in COScells was lower than for the corresponding conditioned medium fromVEGF-A transfected COS cells.

[0134] It was predicted that the mature VEGF-D sequence would be derivedfrom a fragment contained within residues 92-205, with cleavage at FAA^TFY and IIRR^ SIQI. Immunoprecipitation analysis of VEGF-DfullFLAGexpressed in COS cells produced species consistent with the internalproteolytic cleavage of the VEGF-D polypeptide at these sites. Thereforea truncated form of VEGF-D, with the N- and C-terminal regions removed(VEGFDΔNΔC), was produced and expressed in COS cells. This protein wasidentified and purified using the M2 antibody. The VEGFDΔNΔC protein wasalso detected by the A2 antibody, which recognizes a peptide within the92-205 fragment of VEGF-D (not shown). VEGFDΔNΔC was evaluated by theVEGFR2 bioassay and the Miles vascular permeability assay, and shown tobind to and activate the VEGFR2 receptor in a bioassay designed todetect cross-linking of the VEGFR2 extracellular domain. Induction ofvascular permeability by this polypeptide in a Miles assay was at bestmarginal, in contrast to the effect of VEGF-A.

EXAMPLE 10 VEGF-D Binds to and Activates VEGFR-3

[0135] The human VEGF-D cDNA was cloned into baculovirus shuttle vectorsfor the production of reconmbinant VEGF-D. In addition to baculoviralshuttle vectors, which contained the unmodified VEGF-D cDNA (referred toas “full length VEGF-D”) two baculoviral shuttle vectors were assembled,in which the VEGF-D cDNA was modified in the following ways.

[0136] In one construct (referred to as “full length VEGF-D-H₆”) aC-terminal histidine tag was added. In the other construct the putativeN- and C-terminal propeptides were removed, the melittin signal peptidewas fused in-frame to the N-terminus, and a histidine tag was added tothe C-terminus of the remaining VEGF homology domain (referred to as“ΔNΔc-MELsp-VEGF-D-H₆”).

[0137] For each of the three constructs baculoviral clones of two orthree independent transfections were amplified. The supernatant of HighFive (HF) cells was harvested 48 h post infection with high titre virusstocks. The supernatant was adjusted to pH 7 with NaOH and diluted withone volume of D-MEM (0.2% FCS).

[0138] The samples were tested for their ability to stimulate tyrosinephosphorylation of VEGFR-3 (Flt4 receptor) on NIH3T3 cells, as describedby Joukov et al, 1996. The supernatant of uninfected cells and thesupernatant of cells infected with the short splice variant of VEGF-C,which does not stimulate tyrosine phosphorylation of VEGFR-3, were usedas negative controls. VEGF-C modified in the same way asΔNΔC-melSP-VEGF-D-H₆ was used as positive control. The results are shownin FIG. 14.

[0139] The appearance of new bands at 125 and 195 kD indicatesphosphorylation, and hence activation, of the receptor.

EXAMPLE 11 VEGF-D Binds to and Activates VEGFR-2

[0140] Modified and unmodified human VEGF-D cDNA was cloned intobaculovirus shuttle vectors for the production of recombinant VEGF-D asdescribed in Example 10.

[0141] For each of the three constructs full length VEGF-D, full lengthVEGF-D-H₆, and ΔNΔC-melSP-VEGF-D-H₆, baculoviral clones of two or threeindependent transfections were amplified The supernatant of High Five(HF) cells was harvested 48 hours post infection with high titre virusstocks. The supernatant was adjusted to pH 7 with NaOH and diluted withone volume of D-MEM (0.2% FCS).

[0142] The supernatants conditioned with the histidine-tagged proteinswere tested for their ability to stimulate tyrosine phosphorylation ofthe KDR receptor according to Joukov et al, 1996. KDR is the humanhomologue of flk1 (VEGFR-2).

[0143] The supernatant of uninfected cells and the supernatant of cellsinfected with the VEGF-C 156S mutant, which does not stimulate KDR, wereused as negative controls. VEGF₁₆₅ and VEGF-C modified in the same wayas ΔNΔC-melSP-VEGF-D-H₆ were used as positive controls. The results areshown in FIG. 15.

[0144] The appearance of a new band at approximately 210 kD indicatesphosphorylation, and hence activation, of the receptor.

EXAMPLE 12 Analysis of VEGF-D Gene Expression

[0145] In order to characterise the pattern of VEGF-D gene expression inthe human and in mouse embryos, VEGF-D cDNAs were used as hybridizationprobes for Northern blot analysis of polyadenylated human RNA and for insitu hybridization analysis with mouse embryos.

[0146] Gene Expression in the Adult Human

[0147] A 1.1 kb fragment of the human VEGF-D cDNA shown in FIG. 4 (SEQID NO. 4) spanning from the EcoRV site to the 3′-terminus (nucleotides911 to 2029) was labelled with [α-³²P]dATP using the Megaprime DNAlabelling system (Amersham) according to manufacturer's instructions.This probe was used to screen human multiple tissue northern blots(Clontech) by hybridization, also according to manufacturer'sinstructions. These blots contained polyadenylated RNA obtained fromtissues of adult humans who were apparently free of disease.Autoradiography with the labelled blots revealed that VEGF-D mRNA wasmost abundant in heart, lung and skeletal muscle. VEGF-D mRNA was ofintermediate abundance in spleen, ovary, small intestine and colon, andwas of low abundance in kidney, pancreas, thymus, prostate and testis.No VEGF-D mRNA was detected in RNA from brain, placenta, liver orperipheral blood leukocytes. In most of the tissues where VEGF-D mRNAwas detected the size of the transcript was 2.3 kb. The only exceptionwas skeletal muscle, where two VEGF-D transcripts of 2.3 kb and 2.8 kbwere detected. In skeletal muscle the 2.3 kb transcript was moreabundant than the 2.8 kb transcript.

[0148] Gene Expression in Mouse Embryos

[0149] In order to generate an antisense RNA probe for mouse VEGF-DmRNA, the mouse VEGF-D2 cDNA shown in FIG. 7 (SEQ ID NO. 7) was insertedinto the transcription vector pBluescriptIIKS+ (Stratagene). Theresulting plasmid was digested to completion with the restrictionendonuclease FokI and then used as template for an in vitrotranscription reaction with T3 RNA polymerase. This transcriptionreaction gave rise to an antisense RNA probe for VEGF-D mRNA which wascomplementary in sequence to the region of the VEGF-D2 cDNA (FIG. 7)from the 3′-terminus to the FokI cleavage site closest to the3′-terminus (nucleotides 1135 to 700). This antisense RNA probe washybridized at high stringency with paraffin-embedded tissue sectionsgenerated from mouse embryos at post-coital day 15.5. Hybridization andwashing were essentially as described previously (Achen et al., 1995).

[0150] After washing and drying, slides were exposed to autoradiographyfilm for six days.

[0151] Development of the autoradiography film revealed that VEGF-D mRNAis localised in the developing lung of post-coital day 15.5 embryos. Thesignal for VEGF-D mRNA in the lung was strong and highly specific.Control hybridizations with sense probe gave no detectable background inlung or any other tissue.

[0152] Summary

[0153] The VEGF-D gene is broadly expressed in the adult human, but iscertainly not ubiquitously expressed. Strongest expression was detectedin heart, lung and skeletal muscle. In mouse embryos at post-coital day15 5, strong and specific expression of the VEGF-D gene was detected inthe lung. These data suggest that VEGF-D may play a role in lungdevelopment, and that expression of the VEGF-D gene in lung persists inthe adult, at least in humans. Expression of the gene in other tissuesin the adult human suggests that VEGF-D may fulfill other functions inother adult tissues.

EXAMPLE 13 VEGF-D is Mitogenic for Endothelial Cells

[0154] Some members of the VEGF family of proteins, namely VEGF-A (Leunget al, 1989) and VEGF-B (Olofsson et al, 1996), are mitogenic forendothelial cells. In order to test the mitogenic capacity of VEGFDΔNΔCfor endothelial cells, this protein was expressed and purified byaffinity chromatography as described in Example 9. Fraction #3, elutedfrom the M2 affinity column, which contained VEGFDΔNΔC, was diluted 1 in10 in cell culture medium containing 5% serum and applied to bovineaortic endothelial cells (BAEs) which had been propagated in mediumcontaining 10% serum. The BAEs had been seeded in 24-well dishes at adensity of 10,000 cells per well the day before addition of VEGFDΔNΔC,and 3 days after addition of this polypeptide the cells were dissociatedwith trypsin and counted. Purified VEGF-A was included in the experimentas positive control. Results are shown in FIG. 16. The addition offraction #3 to the cell culture medium led to a 2.4-fold increase in thenumber of BAEs after 3 days of incubation, a result which was comparableto that obtained with VEGF-A. Clearly VEGFDΔNΔC is mitogenic forendothelial cells.

EXAMPLE 14 Localization of the VEGF-D Gene on Human Chromosomes

[0155] In order to generate hybridization probes for localization of theVEGF-D gene on human chromosomes, a human genomic DNA clone for VEGF-Dwas isolated from a human genomic DNA library (Clontech). The genomiclibrary was screened by hybridization with the human VEGF-D cDNA shownin FIG. 4, using standard methods (Sambrook et al., 1989). One of theclones thus isolated was shown to contain part of the VEGF-D gene byhybridization to numerous oligonucleotides which were derived insequence from the human VEGF-D cDNA. A region of the genomic clone,approximately 13 kb in size, was purified from agarose gel, labelled bynick-translation with biotin-14-dATP and hybridized in situ at a finalconcentration of 20 ng/μl to metaphases from two normal human males. Thefluorescence in situ hybridization (FISH) method was modified from thatpreviously described (Callen et al, 1990) in that chromosomes werestained before analysis with propidium iodide (as counterstain) and DAPI(for chromosome identification). Images of metaphase preparations werecaptured by a cooled CCD camera, using the CytoVision Ultra imagecollection and enhancement system (Applied Imaging Int. Ltd.). FISHsignals and the DAPI banding pattern were merged for analysis.

[0156] Fifteen metaphases from the first normal male were examined forfluorescent signal. Ten of the metaphases showed signal on one chromatid(3 cells) or both chromatids (7 cells) of the X chromosome in bandp22.1. There was a total of 9 non-specific background dots observed inthese 15 metaphases. A similar result was obtained from hybridization ofthe probe to 15 metaphases from the second normal male, where signal wasobserved at Xp22.1 on one chromatid in 7 cells and on both chromatids in4 cells. In conclusion, the human VEGF-D gene is located on the Xchromosome in band p22.1.

EXAMPLE 15 Localization of the Murine VEGF-D Gene on Mouse Chromosomes

[0157] The mouse chromosomal location of the VEGF-D gene was determinedby interspecific backcross analysis using progeny generated by mating(C57BL/6J×Mus spretus)F1 females and CB7BL/67 males as describedpreviously (Copeland and Jenkins, 1991). This interspecific backcrossmapping panel has been typed for over 2400 loci that are welldistributed among all the autosomes as well as the X chromosome(Copeland and Jenkins, 1991). C57BL/6J and M. spretus DNAs were digestedwith several enzymes and analyzed by Southern blot hybridization forinformative restriction fragment length polymorphisms (RFLPs) using a1.3 kb mouse VEGF-D cDNA probe essentially as described (Jenkins et al.1982). Fragments of 7.1, 6.3, 4.7, 2.5 and 2.2 kb were detected inTaqI-digested C57BL/6J DNA and major fragments of 7.1, 3.7, 2.7 and 2.2kb were detected in TaqI-digested M. spretus DNA. The presence orabsence of the 3.7 and 2.7 TaqI M. spretus-specific fragments, whichcosegregated, was followed in backcross mice. The mapping resultsindicated that the VEGF-D gene is located in the distal region of themouse X chromosome linked to Bik, DxPasI and Ptmb4. Although 89 micewere analyzed for every marker, up to 133 mice were typed for some pairsof markers. Each locus was analyzed in pairwise combinations forrecombination frequencies using the additional data. The ratios of thetotal number of mice exhibiting recombinant chromosomes to the totalnumber of mice analyzed for each pair of loci and the most likely geneorder are: centromere—Btk-14/121-DxPasI-3/99-VEGF-D-5/133-Ptmb4. Therecombination frequencies [expressed as genetic distances incentiMorgans (cM)±the standard error], calculated using Map Manager(version 2.6.5),are—Btk-11.6+/−2.9-DxPasI-3.0+/−1.7-VEGF-D-3.8+/−1.7-Ptmb4. Adescription of the probes and RFLPs for the loci linked to the VEGF-Dgene, including Btk, DxPasI and Ptmb4, has been reported previously(Hacfliger et al., 1992; Holloway et al., 1997).

[0158] We have compared our interspecific map of the X chromosome with acomposite mouse linkage map that reports the map location of manyuncloned mutations (provided from Mouse Genome Database, a computerizeddatabase maintained at The Jackson Library, Bar Harbor, Me.). The VEGF-Dgene mapped in a region of the composite map that lacks mouse mutationswith a phenotype that might be expected for an alteration in the locusfor an endothelial cell mitogen. The distal region of the mouseX-chromosome shares a region of homology with the short arm of the humanX chromosomes (Mouse Genome Database). The placement of the VEGF-D genein this interval in mouse suggests that the human homolog will map toXp22. This is consistent with our FISH analysis which has localized thehuman gene to Xp22.1.

[0159] Numerous disease states are caused by mutations in unknown geneswhich have been mapped to Xp22.1 and the positions immediatelysurrounding this region in the human. These disease states includeKallmann syndrome, ocular albinism (Nettleship-Falls type), ocularalbinism and sensorineural deafness, Partington syndrome,spondyloepiphyseal dysplasia (late), retinitis pigmentosa 15, gonadaldysgenesis (XY female type), Nance-Horan cataract-dental syndrome,retinoschisis, Charcot-Marie-Tooth disease, F-cell production,hypomagnesemia, keratosis follicularis spinulosa decalvans, Coffin-Lowrysyndrome, corneal dermoids, hypophosphatemia, agammaglobulinemia,Aicardi symdrome, hereditary hypophosphatemia II, mental retardation(non-dysmorphic), Opitz G syndrome, pigment disorder (reticulate),dosage-sensitive sex reversal, adrenal hypoplasia, retinitispigmentosa-6, deafness 4 (congenital sensorineural) and Wilson-Turnersyndrome. The positions of the genes involved in these disease statesare documented in the OMIM gene map which is edited by Dr. VictorMcKusick and colleagues at Johns Hopkins University (USA).

[0160] Bioassays to Determine the Function of VEGF-D

[0161] Other assays are conducted to evaluate whether VEGF-D has similaractivities to VEGF in relation to endothelial cell function,angiogenesis and wound healing. Further assays may also be performed,depending on the results of receptor binding distribution studies.

[0162] I. Assays of Endothelial Cell Function

[0163] a) Endothelial Cell Proliferation

[0164] Endothelial cell growth assays are performed by methods wellknown in the art, eg. those of Ferrara & Henzel (1989), Gospodarowicz etal (1989), and/or Claffey et al, Biochim. Biophys. Acta, 1995 1246 1-9.

[0165] b) Cell Adhesion Assay

[0166] The effect of VEGF-D on adhesion of polmorphonuclear granulocytesto endothelial cells is tested.

[0167] c) Chemotaxis

[0168] The standard Boyden chamber chemotaxis assay is used to test theeffect of VEGF-D on chemotaxis.

[0169] d) Plasminogen Activator Assay

[0170] Endothelial cells are tested for the effect of VEGF-D onplasminogen activator and plasminogen activator inhibitor production,using the method of Pepper et al (1991).

[0171] e) Endothelial Cell Migration Assay

[0172] The ability of VEGF-D to stimulate endothelial cells to migrateand form tubes is assayed as described in Montesano et al (1986).Alternatively, the three-dimensional collagen gel assay described byJoukov et al (1996) or a gelatinized membrane in a modified Boydenchamber (Glaser et al, 1980) may be used.

[0173] II Angiogenesis Assay

[0174] The ability of VEGF-D to induce an angiogenic response in chickchorioallantoic membrane is tested as described in Leung et al (1989).Alternatively the rat cornea assay of Rastinejad et al (1989) may beused; this is an accepted method for assay of in vivo angiogenesis, andthe results are readily transferrable to other in vivo systems.

[0175] III Wound Healing

[0176] The ability of VEGF-D to stimulate wound healing is tested in themost clinically relevant model available, as described in Schilling etal (1959) and utilised by Hunt et al (1967).

[0177] IV The Haemopoietic System

[0178] A variety of in vitro and in vivo assays using specific cellpopulations of the haemopoietic system are known in the art, and areoutlined below. In particular a variety of in vitro murine stem cellassays using fluorescence-activated cell sorter purified cells areparticularly convenient:

[0179] a) Repopulating Stem Cells

[0180] These are cells capable of repopulating the bone marrow oflethally irradiated mice, and have the Lin⁻, Rh^(h1), Ly-6A/E⁺, c-kit⁺phenotype. VEGF-D is tested on these cells either alone, or byco-incubation with other factors, followed by measurement of cellularproliferation by ³H-thymidine incorporation.

[0181] b) Late Stage Stem Cells

[0182] These are cells that have comparatively little bone marrowrepopulating ability, but can generate D13 CFU-S. These cells have theLin⁻, Rh^(h1), Ly-6A/E⁺, c-kit⁺ phenotype. VEGF-D is incubated withthese cells for a period of time, injected into lethally irradiatedrecipients, and the number of D13 spleen colonies enumerated.

[0183] c) Progenitor-Enriched Cells

[0184] These are cells that respond in vitro to single growth factorsand have the Lin⁻, Rh^(h1), Ly-6A/E⁺, c-kit⁺ phenotype. This assay willshow if VEGF-D can act directly on haemopoietic progenitor cells. VEGF-Dis incubated with these cells in agar cultures, and the number ofcolonies present after 7-14 days is counted.

[0185] V Atherosclerosis

[0186] Smooth muscle cells play a crucial role in the development orinitiation of atherosclerosis, requiring a change of their phenotypefrom a contractile to a synthetic state. Macrophages, endothelial cells,T lymphocytes and platelets all play a role in the development ofatherosclerotic plaques by influencing the growth and phenotypicmodulations of smooth muscle cell. An in vitro assay using a modifiedRose chamber in which different cell types are seeded on to oppositecoverslips measures the proliferative rate and phenotypic modulations ofsmooth muscle cells in a multicellular environment, and is used toassess the effect of VEGF-D on smooth muscle cells.

[0187] VI Metastasis

[0188] The ability of VEGF-D to inhibit metastasis is assayed using theLewis lung carcinoma model, for example using the method of Cao et al(1995).

[0189] VII VEGF-D in Other Cell Types

[0190] The effects of VEGF-D on proliferation, differentiation andfunction of other cell types, such as liver cells, cardiac muscle andother cells, endocrine cells and osteoblasts can readily be assayed bymethods known in the art, such as ³H-thymidine uptake by in vitrocultures. Expression of VEGF-D in these and other tissues can bemeasured by techniques such as Northern blotting and hybridization or byin situ hybridization.

[0191] VIII Construction of VEGF-D Variants and Analogues

[0192] VEGF-D is a member of the PDGF family of growth factors whichexhibits a high degree of homology to the other members of the PDGFfamily. VEGF-D contains eight conserved cysteine residues which arecharacteristic of this family of growth factors. These conservedcysteine residues form intra-chain disulfide bonds which produce thecysteine knot structure, and inter-chain disulfide bonds that form theprotein dimers which are characteristic of members of the PDGF family ofgrowth factors. VEGF-D will interact with protein tyrosine kinase growthfactor receptors.

[0193] In contrast to proteins where little or nothing is known aboutthe protein structure and active sites needed for receptor binding andconsequent activity, the design of active mutants of VEGF-D is greatlyfacilitated by the fact that a great deal is known about the activesites and important amino acids of the members of the PDGF family ofgrowth factors.

[0194] Published articles elucidating the structure/activityrelationships of members of the PDGF family of growth factors includefor PDGF: Oestman et al, J. Biol. Chem., 1991 266 10073-10077; Anderssonet al, J. Biol. Chem., 1992 267 11260-1266; Oefner et al, EMBO J., 199211 3921-3926; Flemming et al, Molecular and Cell Biol., 1993 134066-4076 and Andersson et al, Growth Factors, 1995 12 159-164; and forVEGF: Kim et al, Growth Factors, 1992 7 53-64; Pötgens et al, J. Biol.Chem., 1994 269 32879-32885 and Claffey et al, Biochem. Biophys. Acta,1995 1246 1-9. From these publications it is apparent that because ofthe eight conserved cysteine residues, the members of the PDGF family ofgrowth factors exhibit a characteristic knotted folding structure anddimerization, which result in formation of three exposed loop regions ateach end of the dimerized molecule, at which the active receptor bindingsites can be expected to be located.

[0195] Based on this information, a person skilled in the biotechnologyarts can design VEGF-D mutants with a very high probability of retainingVEGF-D activity by conserving the eight cysteine residues responsiblefor the knotted folding arrangement and for dimerization, and also byconserving, or making only conservative amino acid substitutions in thelikely receptor sequences in the loop 1, loop 2 and loop 3 region of theprotein structure.

[0196] The formation of desired mutations at specifically targeted sitesin a protein structure is considered to be a standard technique in thearsenal of the protein chemist (Kunkel et al, Methods in Enzymol., 1987154 367-382). Examples of such site-directed mutagenesis with VEGF canbe found in Pötgens et al, J. Biol. Chem., 1994 269 32879-32885 andClaffey et al, Biochim. Biophys. Acta, 1995 1246 1-9. Indeed,site-directed mutagenesis is so common that kits are commerciallyavailable to facilitate such procedures (eg. Promega 1994-1995 Catalog.,Pages 142-145).

[0197] The endothelial cell proliferating activity of VEGF-D mutants canbe readily confirmed by well established screening procedures. Forexample, a procedure analogous to the endothelial cell mitotic assaydescribed by Claffey et al, (Biochim. Biophys. Acta., 1995 1246 1-9) canbe used. Similarly the effects of VEGF-D on proliferation of other celltypes, on cellular differentiation and on human metastasis can be testedusing methods which are well known in the art.

[0198] It will be apparent to the person skilled in the art that whilethe invention has been described in some detail for the purposes ofclarity and understanding, various modifications and alterations to theembodiments and methods described herein may be made without departingfrom the scope of the inventive concept disclosed in this specification.

[0199] References cited herein are listed on the following pages, andare incorporated herein by this reference.

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[0231] Strawn, L. M., McMahon, G., App, H., Schreck, R., Kuchler, W. R.,Longhi, M. P., Hui, T. H., Tang, C., Levitzki, A., Gazit, A., Chen, I.,Keri, G., Orfi, L., Risau, W., Flamme, I., Ullrich, A., Hirth, K. P. andShawver, L. K. Cancer res., 1996 56 3540-3545

1 11 2846 base pairs nucleic acid single linear cDNA NO Human Breast 1GGAATTCAGT GAAGTAAGAA AGACAAAGTG TTCATTGGAG ATTTTTAGTA AGGGGCCAAC 60AGAGCTGCTA AAGTCATGCT TCACTTAACG ATGGGGATAT GTTCGGAGAA ATGCATTGTT 120AGGTGATTTT GTCGTTGTGC AAGCATCTTA GAGTACACTT AGACAAACCT AGCTGGTATA 180ACCTAGGTGT GTAGTAGGAT ATATGGTATA GCCTATTGTT CCTAGGCTAC AAACCCATAC 240AGCATGTTCC TGTACTGAAT ACTGAGGCAA CTGCAACACC GTGGTGAGTA TTTGTGTATC 300TAAACATACC TAAACATAGA AAAGATACAG TAAAAATATG GCATTATAGT CTTATGGGAC 360TACTGTCATA CATACAGTCC ATATATTGTT GACTGTGTAA TGTTGACCTG AATGTCATTA 420TGTGGCAGGC ACATGACTGT GTCGCTAACC TTTGCACAAG ATTACTGTAG GATTACATGA 480GATAGTTGTA AATAATTGGT GGGGTACTGG GCACCTAGTA GGTATGCATA CATGTTCACC 540ATCATTATGG TTGTTTTAAA TCACCTAACC CAGGCCCTGC ACATAGTAAG ACATCAACAA 600ATTGTAGCTG CTACTATTTT GCGCATCTAA TCTTAATATC ATTTATTTTG TAGTCCTTGG 660ATGTTCCCTC CTTTATGACT TCTTTTTTTT TTGTTGTCCT TCCTTTAGCC CTCCATCCTC 720TACAGCTCAG CATCAGAACA CTCTCTTTTT AGACTCCGAT ATGGGGTCCT CCAAGAAAGT 780TACTCTCTCA GTGCTCAGCC GGGAGCAGTC GGAAGGGGTT GGAGCGAGGG TCCGGAGAAG 840CATTGGCAGA CCCGAGTTAA AAAATCTGGA TCCGTTTTTA CTGTTTGATG AATTTAAAGG 900AGGTAGACCA GGAGGATTTC CTGATCATCC ACATCGAGGT TTTGAAACAG TATCCTACCT 960CCTGGAAGGG GGCAGCATGG CCCATGAAGA CTTCTGTGGA CACACTGGTA AAATGAACCC 1020AGGAGATTTG CAGTGGATGA CTGCGGGCCG GGGCATTCTG CACGCTGAGA TGCCTTGCTC 1080AGAGGAGCCA GCCCATGGCC TACAACTGTG GGTTAATTTG AGGAGCTCAG AGAAGATGGT 1140GGAGCCTCAG TACCAGGAAC TGAAAAGTGA AGAAATCCCT AAACCCAGTA AGGATGGTGT 1200GACAGTTGCT GTCATTTCTG GAGAAGCCCT GGGAATAAAG TCCAAGGTTT ACACTCGCAC 1260ACCAACCTTA TATTTGGACT TCAAATTGGA CCCAGGAGCC AAACATTCCC AACCTATCCC 1320TAAAGGGTGG ACAAGCTTCA TTTACACGAT ATCTGGAGAT GTGTATATTG CCCTCTCTAT 1380ATCCCAGCAC AGGTATGCCC AGGGCAGGGT GCCTTTCAGC TTACAGAACA TTCAGTGAGG 1440GAAGAGAATA TGAACACCAG TCATGACACA TCCTGTGCAC AGATGAAAGT CCAGGCACCA 1500TTATGTGTTT TGATACCTCG CTAAGACGTT GGCAACCTCC ATACTGATAA AGGGATGGAG 1560CTACAGTGGA CTCCAAGGGG AGCAGGAATC TGCCTATCTC CTGGGAGAAG GAAATGGAAG 1620GAGGGCCCGA TGATGCACAA CAAAAAATAG AACCTCATCA CACAGCAGTG CTTGGAGAAG 1680GTGACAGTGT CCAAGTGGAG AACAAGGATC CCAAGAGAAG CCACTTTGTC TTAATTGCTG 1740GGGAGCCATT AAGAGAACCA GTTATCCAAC ATGCGATCAT CTCAGTCCAC ATTGGAACGA 1800TCTGAACAGC AGATCAGGGC TGCTTCTAGT TTGGAGGAAC TACTTCGAAT TACTCACTCT 1860GAGGACTGGA AGCTGTGGAG ATGCAGGCTG AGGCTCAAAA GTTTTACCAG TATGGACTCT 1920CGCTCAGCAT CCCATCGGTC CACTAGGTTT GCGGCAACTT TCTATGACAT TGAAACACTA 1980AAAGTTATAG ATGAAGAATG GCAAAGAACT CAGTGCAGCC CTAGAGAAAC GTGCGTGGAG 2040GTGGCCAGTG AGCTGGGGAA GAGTACCAAC ACATTCTTCA AGCCCCCTTG TGTGAACGTG 2100TTCCGATGTG GTGGCTGTTG CAATGAAGAG AGCCTTATCT GTATGAACAC CAGCACCTCG 2160TACATTTCCA AACAGCTCTT TGAGATATCA GTGCCTTTGA CATCAGTACC TGAATTAGTG 2220CCTGTTAAAG TTGCCAATCA TACAGGTTGT AAGTGCTTGC CAACAGCCCC CCGCCATCCA 2280TACTCAATTA TCAGAAGATC CATCCAGATC CCTGAAGAAG ATCGCTGTTC CCATTCCAAG 2340AAACTCTGTC CTATTGACAT GCTATGGGAT AGCAACAAAT GTAAATGTGT TTTGCAGGAG 2400GAAAATCCAC TCGCTGGAAC AGAAGACCAC TCTCATCTCC AGGAACCAGC TCTCTGTGGG 2460CCACACATGA TGTTTGACGA AGATCGTTGC GAGTGTGTCT GTAAAACACC ATGTCCCAAA 2520GATCTAATCC AGCACCCCAA AAACTGCAGT TGCTTTGAGT GCAAAGAAAG TCTGGAGACC 2580TGCTGCCAGA AGCACAAGCT ATTTCACCCA GACACCTGCA GCTGTGAGGA CAGATGCCCC 2640TTTCATACCA GACCATGTGC AAGTGGCAAA ACAGCATGTG CAAAGCATTG CCGCTTTCCA 2700AAGGAGAAAA GGGCTGCCCA GGGGCCCCAC AGCCGAAAGA ATCCTTGATT CAGCGTTCCA 2760AGTTCCCCAT CCCTGTCATT TTTAACAGCA TGCTGCTTTG CCAAGTTGCT GTCACTGTTT 2820TTTTCCCAGG TGTTAAAAAA AAAAAA 2846 13 amino acids amino acid singlelinear peptide NO 2 Pro Xaa Cys Val Xaa Xaa Xaa Arg Cys Xaa Gly Cys Cys1 5 10 325 amino acids amino acid single linear protein NO Human Breast3 Met Arg Ser Ser Gln Ser Thr Leu Glu Arg Ser Glu Gln Gln Ile Arg 1 5 1015 Ala Ala Ser Ser Leu Glu Glu Leu Leu Arg Ile Thr His Ser Glu Asp 20 2530 Trp Lys Leu Trp Arg Cys Arg Leu Arg Leu Lys Ser Phe Thr Ser Met 35 4045 Asp Ser Arg Ser Ala Ser His Arg Ser Thr Arg Phe Ala Ala Thr Phe 50 5560 Tyr Asp Ile Glu Thr Leu Lys Val Ile Asp Glu Glu Trp Gln Arg Thr 65 7075 80 Gln Cys Ser Pro Arg Glu Thr Cys Val Glu Val Ala Ser Glu Leu Gly 8590 95 Lys Ser Thr Asn Thr Phe Phe Lys Pro Pro Cys Val Asn Val Phe Arg100 105 110 Cys Gly Gly Cys Cys Asn Glu Glu Ser Leu Ile Cys Met Asn ThrSer 115 120 125 Thr Ser Tyr Ile Ser Lys Gln Leu Phe Glu Ile Ser Val ProLeu Thr 130 135 140 Ser Val Pro Glu Leu Val Pro Val Lys Val Ala Asn HisThr Gly Cys 145 150 155 160 Lys Cys Leu Pro Thr Ala Pro Arg His Pro TyrSer Ile Ile Arg Arg 165 170 175 Ser Ile Gln Ile Pro Glu Glu Asp Arg CysSer His Ser Lys Lys Leu 180 185 190 Cys Pro Ile Asp Met Leu Trp Asp SerAsn Lys Cys Lys Cys Val Leu 195 200 205 Gln Glu Glu Asn Pro Leu Ala GlyThr Glu Asp His Ser His Leu Gln 210 215 220 Glu Pro Ala Leu Cys Gly ProHis Met Met Phe Asp Glu Asp Arg Cys 225 230 235 240 Glu Cys Val Cys LysThr Pro Cys Pro Lys Asp Leu Ile Gln His Pro 245 250 255 Lys Asn Cys SerCys Phe Glu Cys Lys Glu Ser Leu Glu Thr Cys Cys 260 265 270 Gln Lys HisLys Leu Phe His Pro Asp Thr Cys Ser Cys Glu Asp Arg 275 280 285 Cys ProPhe His Thr Arg Pro Cys Ala Ser Gly Lys Thr Ala Cys Ala 290 295 300 LysHis Cys Arg Phe Pro Lys Glu Lys Arg Ala Ala Gln Gly Pro His 305 310 315320 Ser Arg Lys Asn Pro 325 2029 base pairs nucleic acid single linearcDNA NO Human Lung 4 GTTGGGTTCC AGCTTTCTGT AGCTGTAAGC ATTGGTGGCCACACCACCTC CTTACAAAGC 60 AACTAGAACC TGCGGCATAC ATTGGAGAGA TTTTTTTAATTTTCTGGACA TGAAGTAAAT 120 TTAGAGTGCT TTCTAATTTC AGGTAGAAGA CATGTCCACCTTCTGATTAT TTTTGGAGAA 180 CATTTTGATT TTTTTCATCT CTCTCTCCCC ACCCCTAAGATTGTGCAAAA AAAGCGTACC 240 TTGCCTAATT GAAATAATTT CATTGGATTT TGATCAGAACTGATTATTTG GTTTTCTGTG 300 TGAAGTTTTG AGGTTTCAAA CTTTCCTTCT GGAGAATGCCTTTTGAAACA ATTTTCTCTA 360 GCTGCCTGAT GTCAACTGCT TAGTAATCAG TGGATATTGAAATATTCAAA ATGTACAGAG 420 AGTGGGTAGT GGTGAATGTT TTCATGATGT TGTACGTCCAGCTGGTGCAG GGCTCCAGTA 480 ATGAACATGG ACCAGTGAAG CGATCATCTC AGTCCACATTGGAACGATCT GAACAGCAGA 540 TCAGGGCTGC TTCTAGTTTG GAGGAACTAC TTCGAATTACTCACTCTGAG GACTGGAAGC 600 TGTGGAGATG CAGGCTGAGG CTCAAAAGTT TTACCAGTATGGACTCTCGC TCAGCATCCC 660 ATCGGTCCAC TAGGTTTGCG GCAACTTTCT ATGACATTGAAACACTAAAA GTTATAGATG 720 AAGAATGGCA AAGAACTCAG TGCAGCCCTA GAGAAACGTGCGTGGAGGTG GCCAGTGAGC 780 TGGGGAAGAG TACCAACACA TTCTTCAAGC CCCCTTGTGTGAACGTGTTC CGATGTGGTG 840 GCTGTTGCAA TGAAGAGAGC CTTATCTGTA TGAACACCAGCACCTCGTAC ATTTCCAAAC 900 AGCTCTTTGA GATATCAGTG CCTTTGACAT CAGTACCTGAATTAGTGCCT GTTAAAGTTG 960 CCAATCATAC AGGTTGTAAG TGCTTGCCAA CAGCCCCCCGCCATCCATAC TCAATTATCA 1020 GAAGATCCAT CCAGATCCCT GAAGAAGATC GCTGTTCCCATTCCAAGAAA CTCTGTCCTA 1080 TTGACATGCT ATGGGATAGC AACAAATGTA AATGTGTTTTGCAGGAGGAA AATCCACTTG 1140 CTGGAACAGA AGACCACTCT CATCTCCAGG AACCAGCTCTCTGTGGGCCA CACATGATGT 1200 TTGACGAAGA TCGTTGCGAG TGTGTCTGTA AAACACCATGTCCCAAAGAT CTAATCCAGC 1260 ACCCCAAAAA CTGCAGTTGC TTTGAGTGCA AAGAAAGTCTGGAGACCTGC TGCCAGAAGC 1320 ACAAGCTATT TCACCCAGAC ACCTGCAGCT GTGAGGACAGATGCCCCTTT CATACCAGAC 1380 CATGTGCAAG TGGCAAAACA GCATGTGCAA AGCATTGCCGCTTTCCAAAG GAGAAAAGGG 1440 CTGCCCAGGG GCCCCACAGC CGAAAGAATC CTTGATTCAGCGTTCCAAGT TCCCCATCCC 1500 TGTCATTTTT AACAGCATGC TGCTTTGCCA AGTTGCTGTCACTGTTTTTT TCCCAGGTGT 1560 TAAAAAAAAA ATCCATTTTA CACAGCACCA CAGTGAATCCAGACCAACCT TCCATTCACA 1620 CCAGCTAAGG AGTCCCTGGT TCATTGATGG ATGTCTTCTAGCTGCAGATG CCTCTGCGCA 1680 CCAAGGAATG GAGAGGAGGG GACCCATGTA ATCCTTTTGTTTAGTTTTGT TTTTGTTTTT 1740 TGGTGAATGA GAAAGGTGTG CTGGTCATGG AATGGCAGGTGTCATATGAC TGATTACTCA 1800 GAGCAGATGA GGAAAACTGT AGTCTCTGAG TCCTTTGCTAATCGCAACTC TTGTGAATTA 1860 TTCTGATTCT TTTTTATGCA GAATTTGATT CGTATGATCAGTACTGACTT TCTGATTACT 1920 GTCCAGCTTA TAGTCTTCCA GTTTAATGAA CTACCATCTGATGTTTCATA TTTAAGTGTA 1980 TTTAAAGAAA ATAAACACCA TTATTCAAGC CAAAAAAAAAAAAAAAAAA 2029 354 amino acids amino acid single linear protein NO HumanLung 5 Met Tyr Arg Glu Trp Val Val Val Asn Val Phe Met Met Leu Tyr Val 15 10 15 Gln Leu Val Gln Gly Ser Ser Asn Glu His Gly Pro Val Lys Arg Ser20 25 30 Ser Gln Ser Thr Leu Glu Arg Ser Glu Gln Gln Ile Arg Ala Ala Ser35 40 45 Ser Leu Glu Glu Leu Leu Arg Ile Thr His Ser Glu Asp Trp Lys Leu50 55 60 Trp Arg Cys Arg Leu Arg Leu Lys Ser Phe Thr Ser Met Asp Ser Arg65 70 75 80 Ser Ala Ser His Arg Ser Thr Arg Phe Ala Ala Thr Phe Tyr AspIle 85 90 95 Glu Thr Leu Lys Val Ile Asp Glu Glu Trp Gln Arg Thr Gln CysSer 100 105 110 Pro Arg Glu Thr Cys Val Glu Val Ala Ser Glu Leu Gly LysSer Thr 115 120 125 Asn Thr Phe Phe Lys Pro Pro Cys Val Asn Val Phe ArgCys Gly Gly 130 135 140 Cys Cys Asn Glu Glu Ser Leu Ile Cys Met Asn ThrSer Thr Ser Tyr 145 150 155 160 Ile Ser Lys Gln Leu Phe Glu Ile Ser ValPro Leu Thr Ser Val Pro 165 170 175 Glu Leu Val Pro Val Lys Val Ala AsnHis Thr Gly Cys Lys Cys Leu 180 185 190 Pro Thr Ala Pro Arg His Pro TyrSer Ile Ile Arg Arg Ser Ile Gln 195 200 205 Ile Pro Glu Glu Asp Arg CysSer His Ser Lys Lys Leu Cys Pro Ile 210 215 220 Asp Met Leu Trp Asp SerAsn Lys Cys Lys Cys Val Leu Gln Glu Glu 225 230 235 240 Asn Pro Leu AlaGly Thr Glu Asp His Ser His Leu Gln Glu Pro Ala 245 250 255 Leu Cys GlyPro His Met Met Phe Asp Glu Asp Arg Cys Glu Cys Val 260 265 270 Cys LysThr Pro Cys Pro Lys Asp Leu Ile Gln His Pro Lys Asn Cys 275 280 285 SerCys Phe Glu Cys Lys Glu Ser Leu Glu Thr Cys Cys Gln Lys His 290 295 300Lys Leu Phe His Pro Asp Thr Cys Ser Cys Glu Asp Arg Cys Pro Phe 305 310315 320 His Thr Arg Pro Cys Ala Ser Gly Lys Thr Ala Cys Ala Lys His Cys325 330 335 Arg Phe Pro Lys Glu Lys Arg Ala Ala Gln Gly Pro His Ser ArgLys 340 345 350 Asn Pro 1325 base pairs nucleic acid single linear cDNANO Mouse Lung 6 GGAGAATGCC TTTTGCAACA CTTTTCAGTA GCTGCCTGGA AACAACTGCTTAGTCATCGG 60 TAGACATTTA AAATATTCAA AATGTATGGA GAATGGGGAA TGGGGAATATCCTCATGATG 120 TTCCATGTGT ACTTGGTGCA GGGCTTCAGG AGCGAACATG GACCAGTGAAGGATTTTTCT 180 TTTGAGCGAT CATCCCGGTC CATGTTGGAA CGATCTGAAC AACAGATCCGAGCAGCTTCT 240 AGTTTGGAGG AGTTGCTGCA AATCGCGCAC TCTGAGGACT GGAAGCTGTGGCGATGCCGG 300 TTGAAGCTCA AAAGTCTTGC CAGTATGGAC TCACGCTCAG CATCCCATCGCTCCACCAGA 360 TTTGCGGCAA CTTTCTATGA CACTGAAACA CTAAAAGTTA TAGATGAAGAATGGCAGAGG 420 ACCCAATGCA GCCCTAGAGA GACATGCGTA GAAGTCGCCA GTGAGCTGGGGAAGACAACC 480 AACACATTCT TCAAGCCCCC CTGTGTAAAT GTCTTCCGGT GTGGAGGCTGCTGCAACGAA 540 GAGGGTGTGA TGTGTATGAA CACAAGCACC TCCTACATCT CCAAACAGCTCTTTGAGATA 600 TCAGTGCCTC TGACATCAGT GCCCGAGTTA GTGCCTGTTA AAATTGCCAACCATACGGGT 660 TGTAAGTGCT TGCCCACGGG CCCCCGCCAT CCTTACTCAA TTATCAGAAGATCCATTCAG 720 ACCCCAGAAG AAGATGAATG TCCTCATTCC AAGAAACTCT GTCCTATTGACATGCTGTGG 780 GATAACACCA AATGTAAATG TGTTTTGCAA GACGAGACTC CACTGCCTGGGACAGAAGAC 840 CACTCTTACC TCCAGGAACC CACTCTCTGT GGACCGCACA TGACGTTTGATGAAGATCGC 900 TGTGAGTGCG TCTGTAAAGC ACCATGTCCG GGAGATCTCA TTCAGCACCCGGAAAACTGC 960 AGTTGCTTTG AGTGCAAAGA AAGTCTGGAG AGCTGCTGCC AAAAGCACAAGATTTTTCAC 1020 CCAGACACCT GCAGCTGTGA GGACAGATGT CCTTTTCACA CCAGAACATGTGCAAGTAGA 1080 AAGCCAGCCT GTGGAAAGCA CTGGCGCTTT CCAAAGGAGA CAAGGGCCCAGGGACTCTAC 1140 AGCCAGGAGA ACCCTTGATT CAACTTCCTT TCAAGTCCCC CCATCTCTGTCATTTTAAAC 1200 AGCTCACTGC TTTGTCAAGT TGCTGTCACT GTTGCCCACT ACCCCTTGAACATGTGCAAA 1260 CACAGACACA CACACACACA CACACACAGA GCAACTAGAA TTATGTTTTCTAGGTGCTGC 1320 CTAAG 1325 1135 base pairs nucleic acid single linearcDNA NO Mouse Lung 7 AAACTTTGCT TCTGGAGAAT GCCTTTTGCA ACACTTTTCAGTAGCTGCCT GGAAACAACT 60 GCTTAGTCAT CGGTAGACAT TTAAAATATT CAAAATGTATGGAGAATGGG GAATGGGGAA 120 TATCCTCATG ATGTTCCATG TGTACTTGGT GCAGGGCTTCAGGAGCGAAC ATGGACCAGT 180 GAAGCGATCA TCCCGGTCCA TGTTGGAACG ATCTGAACAACAGATCCGAG CAGCTTCTAG 240 TTTGGAGGAG TTGCTGCAAA TCGCGCACTC TGAGGACTGGAAGCTGTGGC GATGCCGGTT 300 GAAGCTCAAA AGTCTTGCCA GTATGGACTC ACGCTCAGCATCCCATCGCT CCACCAGATT 360 TGCGGCAACT TTCTATGACA CTGAAACACT AAAAGTTATAGATGAAGAAT GGCAGAGGAC 420 CCAATGCAGC CCTAGAGAGA CATGCGTAGA AGTCGCCAGTGAGCTGGGGA AGACAACCAA 480 CACATTCTTC AAGCCCCCCT GTGTAAATGT CTTCCGGTGTGGAGGCTGCT GCAACGAAGA 540 GGGTGTGATG TGTATGAACA CAAGCACCTC CTACATCTCCAAACAGCTCT TTGAGATATC 600 AGTGCCTCTG ACATCAGTGC CCGAGTTAGT GCCTGTTAAAATTGCCAACC ATACGGGTTG 660 TAAGTGCTTG CCCACGGGCC CCCGCCATCC TTACTCAATTATCAGAAGAT CCATTCAGAC 720 CCCAGAAGAA GATGAATGTC CTCATTCCAA GAAACTCTGTCCTATTGACA TGCTGTGGGA 780 TAACACCAAA TGTAAATGTG TTTTGCAAGA CGAGACTCCACTGCCTGGGA CAGAAGACCA 840 CTCTTACCTC CAGGAACCCA CTCTCTGTGG ACCGCACATGACGTTTGATG AAGATCGCTG 900 TGAGTGCGTC TGTAAAGCAC CATGTCCGGG AGATCTCATTCAGCACCCGG AAAACTGCAG 960 TTGCTTTGAG TGCAAAGAAA GTCTGGAGAG CTGCTGCCAAAAGCACAAGA TTTTTCACCC 1020 AGACACCTGC AGGTCAATGG TCTTTTCGCT TTCCCCTTAACTTGGTTTAC TGATGACATT 1080 TAAAGGACAT ACTAATCTGA TCTGTTCAGG CTCTTTTCTCTCAGAGTCCA AGCAC 1135 358 amino acids amino acid single linear proteinMouse Lung 8 Met Tyr Gly Glu Trp Gly Met Gly Asn Ile Leu Met Met Phe HisVal 1 5 10 15 Tyr Leu Val Gln Gly Phe Arg Ser Glu His Gly Pro Val LysAsp Phe 20 25 30 Ser Phe Glu Arg Ser Ser Arg Ser Met Leu Glu Arg Ser GluGln Gln 35 40 45 Ile Arg Ala Ala Ser Ser Leu Glu Glu Leu Leu Gln Ile AlaHis Ser 50 55 60 Glu Asp Trp Lys Leu Trp Arg Cys Arg Leu Lys Leu Lys SerLeu Ala 65 70 75 80 Ser Met Asp Ser Arg Ser Ala Ser His Arg Ser Thr ArgPhe Ala Ala 85 90 95 Thr Phe Tyr Asp Thr Glu Thr Leu Lys Val Ile Asp GluGlu Trp Gln 100 105 110 Arg Thr Gln Cys Ser Pro Arg Glu Thr Cys Val GluVal Ala Ser Glu 115 120 125 Leu Gly Lys Thr Thr Asn Thr Phe Phe Lys ProPro Cys Val Asn Val 130 135 140 Phe Arg Cys Gly Gly Cys Cys Asn Glu GluGly Val Met Cys Met Asn 145 150 155 160 Thr Ser Thr Ser Tyr Ile Ser LysGln Leu Phe Glu Ile Ser Val Pro 165 170 175 Leu Thr Ser Val Pro Glu LeuVal Pro Val Lys Ile Ala Asn His Thr 180 185 190 Gly Cys Lys Cys Leu ProThr Gly Pro Arg His Pro Tyr Ser Ile Ile 195 200 205 Arg Arg Ser Ile GlnThr Pro Glu Glu Asp Glu Cys Pro His Ser Lys 210 215 220 Lys Leu Cys ProIle Asp Met Leu Trp Asp Asn Thr Lys Cys Lys Cys 225 230 235 240 Val LeuGln Asp Glu Thr Pro Leu Pro Gly Thr Glu Asp His Ser Tyr 245 250 255 LeuGln Glu Pro Thr Leu Cys Gly Pro His Met Thr Phe Asp Glu Asp 260 265 270Arg Cys Glu Cys Val Cys Lys Ala Pro Cys Pro Gly Asp Leu Ile Gln 275 280285 His Pro Glu Asn Cys Ser Cys Phe Glu Cys Lys Glu Ser Leu Glu Ser 290295 300 Cys Cys Gln Lys His Lys Ile Phe His Pro Asp Thr Cys Ser Cys Glu305 310 315 320 Asp Arg Cys Pro Phe His Thr Arg Thr Cys Ala Ser Arg LysPro Ala 325 330 335 Cys Gly Lys His Trp Arg Phe Pro Lys Glu Thr Arg AlaGln Gly Leu 340 345 350 Tyr Ser Gln Glu Asn Pro 355 321 amino acidsamino acid single linear protein Mouse Lung 9 Met Tyr Gly Glu Trp GlyMet Gly Asn Ile Leu Met Met Phe His Val 1 5 10 15 Tyr Leu Val Gln GlyPhe Arg Ser Glu His Gly Pro Val Lys Arg Ser 20 25 30 Ser Arg Ser Met LeuGlu Arg Ser Glu Gln Gln Ile Arg Ala Ala Ser 35 40 45 Ser Leu Glu Glu LeuLeu Gln Ile Ala His Ser Glu Asp Trp Lys Leu 50 55 60 Trp Arg Cys Arg LeuLys Leu Lys Ser Leu Ala Ser Met Asp Ser Arg 65 70 75 80 Ser Ala Ser HisArg Ser Thr Arg Phe Ala Ala Thr Phe Tyr Asp Thr 85 90 95 Glu Thr Leu LysVal Ile Asp Glu Glu Trp Gln Arg Thr Gln Cys Ser 100 105 110 Pro Arg GluThr Cys Val Glu Val Ala Ser Glu Leu Gly Lys Thr Thr 115 120 125 Asn ThrPhe Phe Lys Pro Pro Cys Val Asn Val Phe Arg Cys Gly Gly 130 135 140 CysCys Asn Glu Glu Gly Val Met Cys Met Asn Thr Ser Thr Ser Tyr 145 150 155160 Ile Ser Lys Gln Leu Phe Glu Ile Ser Val Pro Leu Thr Ser Val Pro 165170 175 Glu Leu Val Pro Val Lys Ile Ala Asn His Thr Gly Cys Lys Cys Leu180 185 190 Pro Thr Gly Pro Arg His Pro Tyr Ser Ile Ile Arg Arg Ser IleGln 195 200 205 Thr Pro Glu Glu Asp Glu Cys Pro His Ser Lys Lys Leu CysPro Ile 210 215 220 Asp Met Leu Trp Asp Asn Thr Lys Cys Lys Cys Val LeuGln Asp Glu 225 230 235 240 Thr Pro Leu Pro Gly Thr Glu Asp His Ser TyrLeu Gln Glu Pro Thr 245 250 255 Leu Cys Gly Pro His Met Thr Phe Asp GluAsp Arg Cys Glu Cys Val 260 265 270 Cys Lys Ala Pro Cys Pro Gly Asp LeuIle Gln His Pro Glu Asn Cys 275 280 285 Ser Cys Phe Glu Cys Lys Glu SerLeu Glu Ser Cys Cys Gln Lys His 290 295 300 Lys Ile Phe His Pro Asp ThrCys Arg Ser Met Val Phe Ser Leu Ser 305 310 315 320 Pro 20 base pairsnuleic acid single linear oligonucleotide 10 GGGCTGCTTC TAGTTTGGAG 20 20base pairs nuleic acid single linear oligonucleotide 11 CACTCGCAACGATCTTCGTC 20

What is claimed is:
 1. An isolated nucleic acid molecule comprising anucleic acid sequence which encodes a polypeptide comprising a sequenceof amino acids substantially corresponding to the amino acid sequenceset out in SEQ ID NO.3, SEQ ID NO.5, SEQ ID NO.8 or SEQ ID NO. 9, saidpolypeptide having the ability to stimulate vascular permeability orproliferation of endothelial cells, or a fragment or analogue thereofwhich has the ability to stimulate at least one biological activityselected from the group consisting of angiogenesis, vascularpermeability, endothelial cell proliferation, differentiation, migrationor survival, or which has the ability to bind to endothelial cells, butis unable to stimulate at least one of said biological activities.
 2. Anucleic acid molecule according to claim 1, wherein said nucleic acidmolecule comprises a nucleic acid sequence which encodes the amino acidsequence Pro-Xaa-Cys-Val-Xaa-Xaa-Xaa-Arg-Cys-Xaa-Gly-Cys-Cys (SEQ IDNO.2).
 3. A nucleic acid molecule according to claim 1, wherein saidendothelial cells are selected from the group consisting of vascularendothelial cells and lymphatic endothelial cells.
 4. A nucleic acidmolecule according to claim 1, which is a genomic DNA.
 5. A nucleic acidmolecule according to claim 1, which is a cDNA.
 6. A nucleic acidmolecule according to claim 5, which comprises the nucleic acid sequenceof SEQ ID NO.1, SEQ ID NO.4, SEQ ID NO.6, SEQ ID NO.7, or a DNA sequencewhich hybridizes to one of the foregoing sequences under stringentconditions.
 7. A nucleic acid molecule according to claim 6, whichcomprises the nucleic acid sequence of SEQ ID NO.4.
 8. A nucleic acidmolecule according to claim 1, which encodes a polypeptide which has theability to stimulate vascular permeability or proliferation ofendothelial cells.
 9. A nucleic acid molecule according to claim 1,which encodes a polypeptide comprising amino acid residues 64 through172 of SEQ ID NO:3 or amino acid residues 93 through 201 of SEQ ID NO:5.10. A nucleic acid molecule according to claim 9, wherein saidpolypeptide further comprises an affinity tag peptide sequence.
 11. Anucleic acid molecule according to claim 1, which encodes a polypeptidewhich has the ability to bind to endothelial cells but is unable tostimulate endothelial cell proliferation.
 12. A nucleic acid moleculeaccording to claim 11, wherein said endothelial cells are selected fromthe group consisting of vascular endothelial cells and lymphaticendothelial cells.
 13. A nucleic acid molecule according to claim 1,wherein said nucleic acid molecule is a human DNA molecule.
 14. A vectorcomprising a nucleic acid according to claim
 1. 15. A host celltransformed or transformed with a vector according to claim
 14. 16. Anisolated polypeptide which comprises a sequence of amino acidssubstantially corresponding to the amino acid sequence set out in SEQ IDNO.3, SEQ ID NO.5, SEQ ID NO.8 or SEQ ID NO. 9, said polypeptide havingthe ability to stimulate vascular permeability or proliferation ofendothelial cells, or a fragment or analogue thereof which has theability to stimulate at least one endothelial cell biological activityselected from the group consisting of cell proliferation, celldifferentiation, cell migration, cell survival and vascularpermeability, or which has the ability to bind to endothelial cells butis unable to stimulate at least one of said biological activities.
 17. Apolypeptide according to claim 16, wherein said polypeptide comprisesthe amino acid sequencePro-Xaa-Cys-Val-Xaa-Xaa-Xaa-Arg-Cys-Xaa-Gly-Cys-Cys (SEQ ID NO.2).
 18. Apolypeptide according to claim 16, wherein said endothelial cells areselected from the group consisting of vascular endothelial cells andlymphatic endothelial cells.
 19. A polypeptide according to claim 16,which comprises a sequence of amino acids substantially corresponding toSEQ ID NO:3 or SEQ ID NO:5.
 20. A polypeptide according to claim 19,which comprises a sequence of amino acids substantially corresponding toSEQ ID NO
 5. 21. A polypeptide according to claim 16, which has theability to stimulate proliferation of endothelial cells.
 22. Apolypeptide according to claim 16, which has the ability to induceendothelial cell differentiation.
 23. A polypeptide according to claim16, which has the ability to induce vascular permeability.
 24. Apolypeptide according to claim 16, comprising amino acid residues 64through 172 of SEQ ID NO:3 or 93 through 201 of SEQ ID NO:5.
 25. Apolypeptide according to claim 24, further comprising an affinity tagpeptide sequence.
 26. A polypeptide according to claim 16, which has theability to bind to endothelial cells but is unable to stimulateproliferation of endothelial cells.
 27. A polypeptide according to claim26, wherein said endothelial cells are selected from the groupconsisting of vascular endothelial cells and lymphatic endothelialcells.
 28. A polypeptide according to claim 16, wherein said polypeptideis a human protein.
 29. An antibody specifically reactive with apolypeptide according to claim
 16. 30. An antibody according to claim29, wherein said antibody is a polyclonal antibody.
 31. An antibodyaccording to claim 29, wherein said antibody is a monoclonal antibody.32. An antibody according to claim 29, wherein said antibody is labelledwith a detectable label.
 33. A method of making a polypeptide accordingto claim 16, said method comprising the steps of: culturing a host celltransformed or transfected with a vector comprising a nucleic acidsequence encoding said polypeptide operably associated with a promotersequence such that the nucleic acid sequence encoding said polypeptideis expressed; and isolating said polypeptide from said host cell or froma growth medium in which said host cell is cultured.
 34. A method ofisolation of polypeptide according to claim 16, said method comprisingthe step of exposing a cell which expresses said polypeptide to heparinto facilitate release of the polypeptide from the cell, and purifyingthe thus-released polypeptide.
 35. A method of making a vector capableof expressing a polypeptide encoded by a nucleic acid molecule accordingto claim 1, said method comprising inserting said nucleic acid moleculeinto a vector in a position in which said nucleic acid molecule isoperatively connected with at least one promoter.
 36. A vectorcomprising an anti-sense nucleotide sequence, said anti-sense nucleotidesequence being complementary to at least a part of a VEGF-D genomic DNAsequence or a VEGF-D RNA sequence or a cDNA sequence which encodesVEGF-D or a fragment or analogue thereof which promotes at least onebioactivity selected from vascular permeability, proliferation ofendothelial cells and endothelial cell differentiation, whereby saidvector can be used to inhibit said at least one bioactivity.
 37. Amethod of stimulating endothelial cell proliferation comprisingcontacting endothelial cells with an effective endothelial cellproliferation stimulating amount of a polypeptide according to claim 16.38. A method according to claim 37, wherein said endothelial cells areselected from the group consisting of vascular endothelial cells andlymphatic endothelial cells.
 39. A method of stimulating at least onebioactivity selected from endothelial cell proliferation, endothelialcell differentiation and vascular permeability, in vivo in a mammal,said method comprising administering to said mammal an effectivebioactivity stimulating amount of a polypeptide according to claim 16,which has the ability to stimulate said at least one bioactivity.
 40. Amethod according to claim 39, wherein said polypeptide comprises aminoacid residues 64 through 172 of SEQ ID NO:3 or amino acid residues 93through 201 of SEQ ID NO:5.
 41. A method according to claim 39, whereinlymphatic vessel endothelial cell proliferation is stimulated.
 42. Amethod of stimulating at least one bioactivity selected fromangiogenesis and neovascularization in a mammal, said method comprisingthe step of administering to said mammal an effective angiogenesis orneovascularization stimulating amount of a polypeptide according toclaim 16, said polypeptide having the ability to stimulate endothelialcell proliferation.
 43. A method according to claim 42, wherein saidpolypeptide comprises amino acid residues 64 through 172 of SEQ ID NO:3or amino acid residues 93 through 201 of SEQ ID NO:5.
 44. A methodaccording to claim 43, wherein said polypeptide further comprises anaffinity tag peptide sequence.
 45. A method according claim 32, furthercomprising co-administering at least one substance selected from thegroup consisting of VEGF, VEGF-B, VEGF-C, PlGF, PDGF, FGF and heparin.46. A method of inhibiting a bioactivity selected from angiogenesis andneovascularization in a mammal, said method comprising the step ofadministering to said mammal an effective angiogenesis orneovascularization inhibiting amount of a VEGF-D antagonist according toclaim
 70. 47. A method according to claim 46, wherein said VEGF-Dantagonist comprises an antibody specific to VEGF-D.
 48. A methodaccording to claim 46, wherein said VEGF-D antagonist comprises apolypeptide which binds to endothelial cells but which is unable tostimulate at least one biological activity selected from proliferationof endothelial cells, endothelial cell differentiation and vascularpermeability.
 49. A method according to claim 48, wherein saidendothelial cells are selected from the group consisting of vascularendothelial cells and lymphatic endothelial cells.
 50. A method ofinhibiting VEGF-D expression in a mammal comprising the step oftransforming target cells expressing VEGF-D with a vector according toclaim
 36. 51. A pharmaceutical composition comprising a polypeptideaccording to claim 16, and a pharmaceutically acceptable carrier oradjuvant.
 52. A pharmaceutical composition according to claim 51,further comprising at least one substance selected from the groupconsisting of VEGF, VEGF-B, VEGF-C, PlGF, PDGF, FGF and heparin.
 53. Apharmaceutical composition comprising an antibody according to claim 29,and a pharmaceutically acceptable carrier or adjuvant.
 54. Apharmaceutical composition according to claim 53, wherein said antibodyis a monoclonal antibody.
 55. A protein dimer comprising a firstpolypeptide according to claim 16, and a second polypeptide.
 56. Aprotein dimer according to claim 55, wherein said protein dimer is ahomodimer in which the second polypeptide is identical to the firstpolypeptide.
 57. A protein dimer according to claim 55, wherein saidprotein dimer is a heterodimer in which the second polypeptide isselected from VEGF, VEGF-B, VEGF-C, PlGF and PDGF.
 58. A method ofdetecting a polypeptide according to claim 16 in a biological sample,said method comprising the step of contacting the sample with a reagentcapable of binding said polypeptide, and detecting the occurrence ofbinding of said reagent.
 59. A method according to claim 58, whereinsaid reagent comprises an antibody according to claim
 29. 60. A methodof modulating vascular permeability in a mammal, said method comprisingadministering to said mammal an effective vascular permeabilitymodulating amount of a polypeptide according to claim 16 or an antibodythereto.
 61. A method according to claim 60, comprising administering tosaid mammal a polypeptide according to claim 16, having the ability tostimulate endothelial cell proliferation.
 62. A method according toclaim 60, comprising administering to said mammal a polypeptideaccording to claim 16, which has the ability to bind to endothelialcells, but which is unable to stimulate endothelial cell proliferation.63. A method of activation of at least one receptor selected from thegroup consisting of VEGF receptor 2 and VEGF receptor 3, said methodcomprising the step of exposing cells bearing said receptor to aneffective receptor activating dose of a polypeptide according to claim16.
 64. A method according to claim 63, wherein said method is carriedout in vivo.
 65. A method according to claim 63, wherein said method iscarried out in vitro.
 66. A diagnostic or prognostic test kit comprisinga specific binding reagent for a polypeptide according to claim 16, andmeans for detecting binding of said reagent.
 67. A test kit according toclaim 66, wherein said specific binding reagent comprises an antibody tosaid polypeptide.
 68. A diagnostic or prognostic test kit comprising apair of primers specific to a nucleotide sequence according to claim 1,operatively coupled to a polymerase, whereby said polymerase is enabledto selectively amplify the nucleotide sequence from a DNA sample.
 69. Amethod of detecting aberrations in VEGF-D gene structure in a testsubject comprising the steps of: providing a DNA sample from said testsubject; contacting said sample with a set of primers specific to anucleotide sequence according to claim 1, operatively coupled to apolymerase and selectively amplifying said nucleotide sequence from saidsample by polymerase chain reaction; and comparing the nucleotidesequence of the amplified nucleotide sequence from said sample with anucleotide sequence as set forth in SEQ ID NO:1 or SEQ ID NO:4.
 70. AVEGF-D antagonist having the capability to inhibit at least onebiological activity induced by VEGF-D selected from vascularpermeability, endothelial cell proliferation and endothelial celldifferentiation, said antagonist binding to VEGF-D or to a VEGF-Dreceptor, but being less able than VEGF-D to stimulate said at least onebiological activity.
 71. A VEGF-D antagonist according to claim 70,wherein said antagonist comprises an antibody which selectively bindsVEGF-D.
 72. A VEGF-D antagonist according to claim 71, wherein saidantibody is a monoclonal antibody.
 73. A VEGF-D antagonist according toclaim 71, wherein said antagonist comprises a VEGF-D polypeptidefragment or analogue which binds to a VEGF-D receptor, but is less ableto stimulate said at least one biological activity.
 74. A method ofimproving pulmonary blood circulation and/or gas exchange in a mammal,said method comprising administering to said mammal an effective bloodcirculation and/or gas exchange improving amount of a polypeptideaccording to claim
 16. 76. A method of treating fluid accumulation inthe heart and/or lung due to increases in vascular permeability in amammal, said method comprising administering to said mammal an effectivevascular permeability decreasing amount of an antagonist according toclaim
 70. 76. A method of treating an intestinal malabsorption syndromein a patient suffering therefrom, said method comprising administeringto said patient an effective intestinal blood circulation and/orvascular permeability increasing amount of a polypeptide according toclaim 16.